Publications by authors named "Stefano Pluchino"

106 Publications

Stem Cell Therapies for Progressive Multiple Sclerosis.

Front Cell Dev Biol 2021 9;9:696434. Epub 2021 Jul 9.

Department of Clinical Neurosciences and National Institute for Health Research (NIHR) Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal degeneration. MS patients typically present with a relapsing-remitting (RR) disease course, manifesting as sporadic attacks of neurological symptoms including ataxia, fatigue, and sensory impairment. While there are several effective disease-modifying therapies able to address the inflammatory relapses associated with RRMS, most patients will inevitably advance to a progressive disease course marked by a gradual and irreversible accrual of disabilities. Therapeutic intervention in progressive MS (PMS) suffers from a lack of well-characterized biological targets and, hence, a dearth of successful drugs. The few medications approved for the treatment of PMS are typically limited in their efficacy to forms of the disease, have little impact on slowing degeneration, and fail to promote repair. In looking to address these unmet needs, the multifactorial therapeutic benefits of stem cell therapies are particularly compelling. Ostensibly providing neurotrophic support, immunomodulation and cell replacement, stem cell transplantation holds substantial promise in combatting the complex pathology of chronic neuroinflammation. Herein, we explore the current state of preclinical and clinical evidence supporting the use of stem cells in treating PMS and we discuss prospective hurdles impeding their translation into revolutionary regenerative medicines.
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http://dx.doi.org/10.3389/fcell.2021.696434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8299560PMC
July 2021

Metabolic Control of Smoldering Neuroinflammation.

Front Immunol 2021 23;12:705920. Epub 2021 Jun 23.

Department of Clinical Neurosciences and National Institute for Health Research (NIHR) Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom.

Compelling evidence exists that patients with chronic neurological conditions, which includes progressive multiple sclerosis, display pathological changes in neural metabolism and mitochondrial function. However, it is unknown if a similar degree of metabolic dysfunction occurs also in non-neural cells in the central nervous system. Specifically, it remains to be clarified (i) the full extent of metabolic changes in tissue-resident microglia and infiltrating macrophages after prolonged neuroinflammation (e.g., at the level of chronic active lesions), and (ii) whether these alterations underlie a unique pathogenic phenotype that is amenable for therapeutic targeting. Herein, we discuss how cell metabolism and mitochondrial function govern the function of chronic active microglia and macrophages brain infiltrates and identify new metabolic targets for therapeutic approaches aimed at reducing smoldering neuroinflammation.
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http://dx.doi.org/10.3389/fimmu.2021.705920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262770PMC
June 2021

Physiological and Pathological Factors Affecting Drug Delivery to the Brain by Nanoparticles.

Adv Sci (Weinh) 2021 06 15;8(11):e2002085. Epub 2021 Mar 15.

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK.

The prevalence of neurological/neurodegenerative diseases, such as Alzheimer's disease is known to be increasing due to an aging population and is anticipated to further grow in the decades ahead. The treatment of brain diseases is challenging partly due to the inaccessibility of therapeutic agents to the brain. An increasingly important observation is that the physiology of the brain alters during many brain diseases, and aging adds even more to the complexity of the disease. There is a notion that the permeability of the blood-brain barrier (BBB) increases with aging or disease, however, the body has a defense mechanism that still retains the separation of the brain from harmful chemicals in the blood. This makes drug delivery to the diseased brain, even more challenging and complex task. Here, the physiological changes to the diseased brain and aged brain are covered in the context of drug delivery to the brain using nanoparticles. Also, recent and novel approaches are discussed for the delivery of therapeutic agents to the diseased brain using nanoparticle based or magnetic resonance imaging guided systems. Furthermore, the complement activation, toxicity, and immunogenicity of brain targeting nanoparticles as well as novel in vitro BBB models are discussed.
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http://dx.doi.org/10.1002/advs.202002085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188209PMC
June 2021

Neural stem cells traffic functional mitochondria via extracellular vesicles.

PLoS Biol 2021 04 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

NSCs: Sentinel Cells of the Brain.

Cell Stem Cell 2021 02;28(2):177-179

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

Adult neural stem cells (NSCs) have the ability to oscillate between activated and dormant states in response to extrinsic regulators. In this issue of Cell Stem Cell, Belenguer et al. (2020) identify a direct role for systemic TNF-α, which acts through its receptors TNFRII and TNFRI as a regulator of NSC activation and a return to quiescence, respectively.
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http://dx.doi.org/10.1016/j.stem.2020.11.016DOI Listing
February 2021

The Inclusion of a Matrix Metalloproteinase-9 Responsive Sequence in Self-assembled Peptide-based Brain-Targeting Nanoparticles Improves the Efficiency of Nanoparticles Crossing the Blood-Brain Barrier at Elevated MMP-9 Levels.

J Pharm Sci 2021 03 15;110(3):1349-1364. Epub 2020 Dec 15.

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK. Electronic address:

This study investigated whether the inclusion of a matrix metalloproteinase-9 (MMP-9) responsive sequence in self-assembled peptide-based brain-targeting nanoparticles (NPs) would enhance the blood-brain barrier (BBB) penetration when MMP-9 levels are elevated both in the brain and blood circulation. Brain-targeting peptides were conjugated at the N-terminus to MMP-9-responsive peptides, and these were conjugated at the N-terminus to lipid moiety (cholesteryl chloroformate or palmitic acid). Two constructs did not have MMP-9-responsive peptides. NPs were characterised for size, charge, critical micelle concentration, toxicity, blood compatibility, neural cell uptake, release profiles, and in vitro BBB permeability simulating normal or elevated MMP-9 levels. The inclusion of MMP-9-sensitive sequences did not improve the release of a model drug in the presence of active MMP-9 from NPs compared to distilled water. F NMR studies suggested the burial of MMP-9-sensitive sequences inside the NPs making them inaccessible to MMP-9. Only cholesterol-GGGCKAPETALC (responsive to MMP-9) NPs showed <5% haemolysis, <1 pg/mL release of IL-1β at 500 μg/mL from THP1 cells, with 70.75 ± 5.78% of NPs crossing the BBB at 24 h in presence of active MMP-9. In conclusion, brain-targeting NPs showed higher transport across the BBB model when MMP-9 levels were elevated and the brain-targeting ligand was responsive to MMP-9.
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http://dx.doi.org/10.1016/j.xphs.2020.12.004DOI Listing
March 2021

Succinate Receptor 1: An Emerging Regulator of Myeloid Cell Function in Inflammation.

Trends Immunol 2021 01 2;42(1):45-58. Epub 2020 Dec 2.

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

The rapidly evolving area of immunometabolism has shed new light on the fundamental properties of products and intermediates of cellular metabolism (metabolites), highlighting their key signaling roles in cell-to-cell communication. Recent evidence identifies the succinate-succinate receptor 1 (SUCNR1) axis as an essential regulator of tissue homeostasis. Succinate signaling via SUCNR1 guides divergent responses in immune cells, which are tissue and context dependent. Herein, we explore the main cellular pathways regulated by the succinate-SUCNR1 axis and focus on the biology of SUCNR1 and its roles influencing the function of myeloid cells. Hence, we identify new therapeutic targets and putative therapeutic approaches aimed at resolving detrimental myeloid cell responses in tissues, including those occurring in the persistently inflamed central nervous system (CNS).
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http://dx.doi.org/10.1016/j.it.2020.11.004DOI Listing
January 2021

Harnessing the Neural Stem Cell Secretome for Regenerative Neuroimmunology.

Front Cell Neurosci 2020 5;14:590960. Epub 2020 Nov 5.

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

Increasing evidence foresees the of neural stem cells (NSCs) to confer superimposable beneficial properties as exogenous NSC transplants in experimental treatments of traumas and diseases of the central nervous system (CNS). Naturally produced biologics include membrane-free signaling molecules and extracellular membrane vesicles (EVs) capable of regulating broad functional responses. The development of high-throughput screening pipelines for the identification and validation of NSC targets is still in early development. Encouraging results from pre-clinical animal models of disease have highlighted -based (acellular) therapeutics as providing significant improvements in biochemical and behavioral measurements. Most of these responses are being hypothesized to be the result of modulating and promoting the restoration of key inflammatory and regenerative programs in the CNS. Here, we will review the most recent findings regarding the identification of NSC-secreted factors capable of modulating the immune response to promote the regeneration of the CNS in animal models of CNS trauma and inflammatory disease and discuss the increased interest to refine the pro-regenerative features of the NSC into a clinically available therapy in the emerging field of Regenerative Neuroimmunology.
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http://dx.doi.org/10.3389/fncel.2020.590960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674923PMC
November 2020

Systematic approach to selecting licensed drugs for repurposing in the treatment of progressive multiple sclerosis.

J Neurol Neurosurg Psychiatry 2021 Mar 12;92(3):295-302. Epub 2020 Nov 12.

Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK.

Objective: To establish a rigorous, expert-led, evidence-based approach to the evaluation of licensed drugs for repurposing and testing in clinical trials of people with progressive multiple sclerosis (MS).

Methods: We long-listed licensed drugs with evidence of human safety, blood-brain barrier penetrance and demonstrable efficacy in at least one animal model, or mechanistic target, agreed by a panel of experts and people with MS to be relevant to the pathogenesis of progression. We systematically reviewed the preclinical and clinical literature for each compound, condensed this into a database of summary documents and short-listed drugs by scoring each one of them. Drugs were evaluated for immediate use in a clinical trial, and our selection was scrutinised by a final independent expert review.

Results: From a short list of 55 treatments, we recommended four treatments for immediate testing in progressive MS: R-α-lipoic acid, metformin, the combination treatment of R-α-lipoic acid and metformin, and niacin. We also prioritised clemastine, lamotrigine, oxcarbazepine, nimodipine and flunarizine.

Conclusions: We report a standardised approach for the identification of candidate drugs for repurposing in the treatment of progressive MS.
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http://dx.doi.org/10.1136/jnnp-2020-324286DOI Listing
March 2021

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

Stem Cells of the Aging Brain.

Front Aging Neurosci 2020 6;12:247. Epub 2020 Aug 6.

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

The adult central nervous system (CNS) contains resident stem cells within specific niches that maintain a self-renewal and proliferative capacity to generate new neurons, astrocytes, and oligodendrocytes throughout adulthood. Physiological aging is associated with a progressive loss of function and a decline in the self-renewal and regenerative capacities of CNS stem cells. Also, the biggest risk factor for neurodegenerative diseases is age, and current and models of neurodegenerative diseases rarely consider this. Therefore, combining both aging research and appropriate interrogation of animal disease models towards the understanding of the disease and age-related stem cell failure is imperative to the discovery of new therapies. This review article will highlight the main intrinsic and extrinsic regulators of neural stem cell (NSC) aging and discuss how these factors impact normal homeostatic functions within the adult brain. We will consider established animal and human disease model systems, and then discuss the current and future trajectories of novel senotherapeutics that target aging NSCs to ameliorate brain disease.
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http://dx.doi.org/10.3389/fnagi.2020.00247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426063PMC
August 2020

Development of Brain Targeting Peptide Based MMP-9 Inhibiting Nanoparticles for the Treatment of Brain Diseases with Elevated MMP-9 Activity.

J Pharm Sci 2020 10 2;109(10):3134-3144. Epub 2020 Jul 2.

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK. Electronic address:

Latent and active levels of cerebral matrix metalloproteinase 9 (MMP-9) are elevated in neurological diseases and brain injuries, contributing to neurological damage and poor clinical outcomes. This study aimed developing peptide-based nanoparticles with ability to cross the blood-brain-barrier (BBB) and inhibit MMP-9. Three amphiphilic peptides were synthesised containing brain-targeting ligands (HAIYPRH or CKAPETALC) conjugated with MMP-9 inhibiting peptide (CTTHWGFTLC) linked by glycine (spacer) at the N-terminus, and the peptide sequences were conjugated at the N- terminus to cholesterol. F NMR assay was developed to measure MMP-9 inhibition. Cell toxicity was evaluated by the LDH assay, and dialysis studies were conducted with/without fetal bovine serum. An in vitro model was employed to evaluate the ability of nanoparticles crossing the BBB. The amphiphilic peptide (Cholesterol-GGGCTTHWGFTLCHAIYPRH) formed nanoparticles (average size of 202.8 nm) with ability to cross the BBB model. MMP-9 inhibiting nanoparticles were non-toxic to cells, and reduced MMP-9 activity from kobs of 4.5 × 10s to complete inhibition. Dialysis studies showed that nanoparticles did not disassemble by extreme dilution (40 folds), but gradually hydrolysed by serum enzymes. In conclusion, the MMP-9 inhibiting nanoparticles reduced the activity of MMP-9, with acceptable serum stability, minimal cell toxicity and ability to cross the in vitro BBB model.
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http://dx.doi.org/10.1016/j.xphs.2020.06.021DOI Listing
October 2020

Transplantation of induced neural stem cells (iNSCs) into chronically demyelinated corpus callosum ameliorates motor deficits.

Acta Neuropathol Commun 2020 06 9;8(1):84. Epub 2020 Jun 9.

Department of Anatomy, Physiology and Genetics, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd, Bethesda, MD, 20814, USA.

Multiple Sclerosis (MS) causes neurologic disability due to inflammation, demyelination, and neurodegeneration. Immunosuppressive treatments can modify the disease course but do not effectively promote remyelination or prevent long term neurodegeneration. As a novel approach to mitigate chronic stage pathology, we tested transplantation of mouse induced neural stem cells (iNSCs) into the chronically demyelinated corpus callosum (CC) in adult mice. Male C57BL/6 mice fed 0.3% cuprizone for 12 weeks exhibited CC atrophy with chronic demyelination, astrogliosis, and microglial activation. Syngeneic iNSCs were transplanted into the CC after ending cuprizone and perfused for neuropathology 2 weeks later. Magnetic resonance imaging (MRI) sequences for magnetization transfer ratio (MTR), diffusion-weighted imaging (T2), and diffusion tensor imaging (DTI) quantified CC pathology in live mice before and after iNSC transplantation. Each MRI technique detected progressive CC pathology. Mice that received iNSCs had normalized DTI radial diffusivity, and reduced astrogliosis post-imaging. A motor skill task that engages the CC is Miss-step wheel running, which demonstrated functional deficits from cuprizone demyelination. Transplantation of iNSCs resulted in marked recovery of running velocity. Neuropathology after wheel running showed that iNSC grafts significantly increased host oligodendrocytes and proliferating oligodendrocyte progenitors, while modulating axon damage. Transplanted iNSCs differentiated along astrocyte and oligodendrocyte lineages, without myelinating, and many remained neural stem cells. Our findings demonstrate the applicability of neuroimaging and functional assessments for pre-clinical interventional trials during chronic demyelination and detect improved function from iNSC transplantation. Directly reprogramming fibroblasts into iNSCs facilitates the future translation towards exogenous autologous cell therapies.
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http://dx.doi.org/10.1186/s40478-020-00960-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285785PMC
June 2020

Promises and Limitations of Neural Stem Cell Therapies for Progressive Multiple Sclerosis.

Trends Mol Med 2020 10 21;26(10):898-912. Epub 2020 May 21.

Department of Clinical Neurosciences and National Institute for Health Research (NIHR) Biomedical Research Centre, University of Cambridge, Cambridge, UK.

Multiple disease-modifying medications with regulatory approval to treat multiple sclerosis (MS) are unable to prevent inflammatory tissue damage in the central nervous system (CNS), and none directly promote repair. Thus, there is an unmet clinical need for therapies that can arrest and reverse the persistent accumulation of disabilities associated with progressive forms of MS (P-MS). Preclinical research has revealed an unexpected ability of neural stem cell (NSC) therapies to provide neurotrophic support and inhibit detrimental host immune responses in vivo following transplantation into the chronically inflamed CNS. We discuss NSC transplantation as a promising therapy for P-MS, elaborate on the necessities of clinical trial validation and formalized usage guidelines, and caution about unscrupulous 'clinics' marketing unproven therapies to patients.
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http://dx.doi.org/10.1016/j.molmed.2020.04.005DOI Listing
October 2020

Characterization of Human iPSC-derived Spinal Motor Neurons by Single-cell RNA Sequencing.

Neuroscience 2020 12 5;450:57-70. Epub 2020 May 5.

Department of Neurology and Neurosurgery, Montreal Neurological Institute-Hospital, McGill University, 3801, rue University, Montreal (Quebec) H3A 2B4, Canada. Electronic address:

Human induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, with a number of molecularly distinct subpopulations present within this MN group. Roughly 19% of induced MNs resemble hypaxial motor column MNs, while ∼6% of induced MNs have features of median motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and disease.
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http://dx.doi.org/10.1016/j.neuroscience.2020.04.041DOI Listing
December 2020

Intrinsic antiviral immunity drives neurodegeneration in Alzheimer disease.

J Clin Invest 2020 04;130(4):1622-1624

β-Amyloid aggregates found in brain plaques are viewed as triggers of cytotoxicity and neuroinflammation in Alzheimer disease (AD). However, the main β-amyloid (Aβ) species and what imbues the aggregates with such toxic potential are still not yet understood. In this issue of the JCI, Roy et al. show that Aβ complexed with nucleic acids triggers an antiviral type I interferon response in neuroglia, resulting in complement-mediated synapse elimination in AD models. These findings identify a putative endogenous immune signaling axis that drives neurodegeneration in AD and has strong implications for the development of precise therapeutic strategies.
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http://dx.doi.org/10.1172/JCI135906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108882PMC
April 2020

Refining rodent models of spinal cord injury.

Exp Neurol 2020 06 3;328:113273. Epub 2020 Mar 3.

Centre for Neuroscience, Surgery and Trauma, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark St, London E1 2AT, UK. Electronic address:

This report was produced by an Expert Working Group (EWG) consisting of UK-based researchers, veterinarians and regulators of animal experiments with specialist knowledge of the use of animal models of spinal cord injury (SCI). It aims to facilitate the implementation of the Three Rs (Replacement, Reduction and Refinement), with an emphasis on refinement. Specific animal welfare issues were identified and discussed, and practical measures proposed, with the aim of reducing animal use and suffering, reducing experimental variability, and increasing translatability within this critically important research field.
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http://dx.doi.org/10.1016/j.expneurol.2020.113273DOI Listing
June 2020

Parkinson's disease, aging and adult neurogenesis: Wnt/β-catenin signalling as the key to unlock the mystery of endogenous brain repair.

Aging Cell 2020 03 12;19(3):e13101. Epub 2020 Feb 12.

Department of Biomedical and Biotechnological Sciences (BIOMETEC), Pharmacology and Physiology Sections, Medical School, University of Catania, Catania, Italy.

A common hallmark of age-dependent neurodegenerative diseases is an impairment of adult neurogenesis. Wingless-type mouse mammary tumor virus integration site (Wnt)/β-catenin (WβC) signalling is a vital pathway for dopaminergic (DAergic) neurogenesis and an essential signalling system during embryonic development and aging, the most critical risk factor for Parkinson's disease (PD). To date, there is no known cause or cure for PD. Here we focus on the potential to reawaken the impaired neurogenic niches to rejuvenate and repair the aged PD brain. Specifically, we highlight WβC-signalling in the plasticity of the subventricular zone (SVZ), the largest germinal region in the mature brain innervated by nigrostriatal DAergic terminals, and the mesencephalic aqueduct-periventricular region (Aq-PVR) Wnt-sensitive niche, which is in proximity to the SNpc and harbors neural stem progenitor cells (NSCs) with DAergic potential. The hallmark of the WβC pathway is the cytosolic accumulation of β-catenin, which enters the nucleus and associates with T cell factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors, leading to the transcription of Wnt target genes. Here, we underscore the dynamic interplay between DAergic innervation and astroglial-derived factors regulating WβC-dependent transcription of key genes orchestrating NSC proliferation, survival, migration and differentiation. Aging, inflammation and oxidative stress synergize with neurotoxin exposure in "turning off" the WβC neurogenic switch via down-regulation of the nuclear factor erythroid-2-related factor 2/Wnt-regulated signalosome, a key player in the maintenance of antioxidant self-defense mechanisms and NSC homeostasis. Harnessing WβC-signalling in the aged PD brain can thus restore neurogenesis, rejuvenate the microenvironment, and promote neurorescue and regeneration.
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http://dx.doi.org/10.1111/acel.13101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059166PMC
March 2020

The neural stem cell secretome and its role in brain repair.

Brain Res 2020 02 19;1729:146615. Epub 2019 Dec 19.

Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, Hills Road, CB2 0HA Cambridge, UK.

Compelling evidence from experimental animal disease models and early-phase clinical trials identifies the transplantation of neural progenitor/stem cells (NSCs) as a viable path towards the development of clinically applicable exogenous stem cell therapies. Building from current advances in the field of NSC biology and following the positive outcomes of NSC transplantation studies, the contemporary view is that transplanted NSCs act as local 'factories' capable of producing and secreting a wide array of immune and neurotrophic factors. This has launched a 'stem cell race' to identify the mechanisms behind stem-cell mediated repair in what has been labeled the paracrine hypothesis. This hypothesis proposes that NSC grafts act as a natural source of potent biologics capable of modulating and promoting the restoration of several key functions in the central nervous system (CNS) tissue following acute or chronic tissue damage. Investigators have been inspired to examine novel ways to harness and utilize the pro-regenerative properties of NSC therapies as an alternative approach to a more classical (small molecule based) treatment of CNS diseases. In this review, we will discuss the most recent findings of human NSC (hNSCs) transplants in experimental animal models of CNS diseases that identify of hNSC-secreted factors, including those trafficked within extracellular membrane vesicles (EVs), and the outcomes of recent clinical trials utilizing hNSC therapeutics in CNS diseases.
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http://dx.doi.org/10.1016/j.brainres.2019.146615DOI Listing
February 2020

Explicating Exosomes: Reclassifying the Rising Stars of Intercellular Communication.

Cell 2019 04;177(2):225-227

Cambridge Innovation Technologies Consulting (CITC) Limited, UK.

There is growing interest surrounding the diagnostic and therapeutic potential of exosomes, but a definitive description of these extracellular vesicles remains elusive. In this issue, Jeppesen et al. characterize exosomes following a strict isolation protocol and in so doing challenge several of the accepted properties of these agents of intercellular communication.
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http://dx.doi.org/10.1016/j.cell.2019.03.020DOI Listing
April 2019

SUMOylation promotes survival and integration of neural stem cell grafts in ischemic stroke.

EBioMedicine 2019 Apr 21;42:214-224. Epub 2019 Mar 21.

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

Background: Neural stem cell (NSC)-based therapies hold great promise for treating diseases of the central nervous system (CNS). However, several fundamental problems still need to be overcome to fully exploit the clinical potential of NSC therapeutics. Chief among them is the limited survival of NSC grafts within hostile microenvironments.

Methods: Herein, we sought to engineer NSCs in an effort to increase graft survival within ischemic brain lesions via upregulation of global SUMOylation, a post-translational modification critically involved in mediating tolerance to ischemia/reperfusion.

Findings: NSCs overexpressing the SUMO E2-conjugase Ubc9 displayed resistance to oxygen-glucose-deprivation/restoration of oxygen/glucose (OGD/ROG) and enhanced neuronal differentiation in vitro, as well as increased survival and neuronal differentiation when transplanted in mice with transient middle cerebral artery occlusion in vivo.

Interpretation: Our work highlights a critical role for SUMOylation in NSC biology and identifies a biological pathway that can be targeted to increase the effectiveness of exogenous stem cell medicines in ischemic stroke. FUND: Intramural Research Program of the NINDS/NIH, the Italian Multiple Sclerosis Foundation (FISM), the Bascule Charitable Trust, NIH-IRTA-OxCam and Wellcome Trust Research Training Fellowships.
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http://dx.doi.org/10.1016/j.ebiom.2019.03.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491415PMC
April 2019

Foxg1 Antagonizes Neocortical Stem Cell Progression to Astrogenesis.

Cereb Cortex 2019 12;29(12):4903-4918

Laboratory of Cerebral Cortex Development, Neuroscience Area, SISSA, Trieste, Italy.

Neocortical astrogenesis follows neuronogenesis and precedes oligogenesis. Among key factors dictating its temporal articulation, there are progression rates of pallial stem cells (SCs) towards astroglial lineages as well as activation rates of astrocyte differentiation programs in response to extrinsic gliogenic cues. In this study, we showed that high Foxg1 SC expression antagonizes astrocyte generation, while stimulating SC self-renewal and committing SCs to neuronogenesis. We found that mechanisms underlying this activity are mainly cell autonomous and highly pleiotropic. They include a concerted downregulation of 4 key effectors channeling neural SCs to astroglial fates, as well as defective activation of core molecular machineries implementing astroglial differentiation programs. Next, we found that SC Foxg1 levels specifically decline during the neuronogenic-to-gliogenic transition, pointing to a pivotal Foxg1 role in temporal modulation of astrogenesis. Finally, we showed that Foxg1 inhibits astrogenesis from human neocortical precursors, suggesting that this is an evolutionarily ancient trait.
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http://dx.doi.org/10.1093/cercor/bhz031DOI Listing
December 2019

Targeting Mitochondrial Metabolism in Neuroinflammation: Towards a Therapy for Progressive Multiple Sclerosis.

Trends Mol Med 2018 10 9;24(10):838-855. Epub 2018 Aug 9.

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

The lack of effective treatment options for chronic neurological conditions, such as multiple sclerosis (MS), highlights the need to re-evaluate disease pathophysiology in the process of identifying novel therapeutic targets. The persistent activation of mononuclear phagocytes (MPs) is one of the major drivers of neurodegeneration and it sustains central nervous system (CNS) damage. Mitochondrial metabolism influences the activity of MPs, and the metabolites that they produce have key signalling roles in inflammation. However, how changes in immune cell metabolism sustain a chronic state of neuroinflammation is not fully understood. Novel molecular and cellular therapies for chronic neuroinflammation should be developed to target mitochondrial metabolism in innate immune cells to prevent secondary neurological damage and the accumulation of irreversible disability in patients.
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http://dx.doi.org/10.1016/j.molmed.2018.07.007DOI Listing
October 2018

Neural Stem Cell Grafts Promote Astroglia-Driven Neurorestoration in the Aged Parkinsonian Brain via Wnt/β-Catenin Signaling.

Stem Cells 2018 08 16;36(8):1179-1197. Epub 2018 Apr 16.

Oasi Research Institute-IRCCS, Troina, Italy.

During aging-one the most potent risk factors for Parkinson's disease (PD)-both astrocytes and microglia undergo functional changes that ultimately hamper homoeostasis, defense, and repair of substantia nigra pars compacta (SNpc) midbrain dopaminergic (mDA) neurons. We tested the possibility of rejuvenating the host microenvironment and boosting SNpc DA neuronal plasticity via the unilateral transplantation of syngeneic neural stem/progenitor cells (NSCs) in the SNpc of aged mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced experimental PD. Transplanted NSCs within the aged SNpc engrafted and migrated in large proportions to the tegmental aqueduct mDA niche, with 30% acquiring an astroglial phenotype. Both graft-derived exogenous (ex-Astro) and endogenous astrocytes (en-Astro) expressed Wnt1. Both ex-Astro and en-Astro were key triggers of Wnt/β-catenin signaling in SNpc-mDA neurons and microglia, which was associated with mDA neurorescue and immunomodulation. At the aqueduct-ventral tegmental area level, NSC grafts recapitulated a genetic Wnt1-dependent mDA developmental program, inciting the acquisition of a mature Nurr1 TH neuronal phenotype. Wnt/β-catenin signaling antagonism abolished mDA neurorestoration and immune modulatory effects of NSC grafts. Our work implicates an unprecedented therapeutic potential for somatic NSC grafts in the restoration of mDA neuronal function in the aged Parkinsonian brain. Stem Cells 2018;36:1179-1197.
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http://dx.doi.org/10.1002/stem.2827DOI Listing
August 2018

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

Genome Biol 2018 03 15;19(1):32. Epub 2018 Mar 15.

The Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.

Background: The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality.

Results: We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers.

Conclusions: This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.
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http://dx.doi.org/10.1186/s13059-018-1405-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853149PMC
March 2018

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

Microglia Polarization, Gene-Environment Interactions and Wnt/β-Catenin Signaling: Emerging Roles of Glia-Neuron and Glia-Stem/Neuroprogenitor Crosstalk for Dopaminergic Neurorestoration in Aged Parkinsonian Brain.

Front Aging Neurosci 2018 12;10:12. Epub 2018 Feb 12.

Oasi ResearchInstitute-IRCCS, Troina, Italy.

Neuroinflammatory processes are recognized key contributory factors in Parkinson's disease (PD) physiopathology. While the causes responsible for the progressive loss of midbrain dopaminergic (mDA) neuronal cell bodies in the subtantia nigra pars compacta are poorly understood, aging, genetics, environmental toxicity, and particularly inflammation, represent prominent etiological factors in PD development. Especially, reactive astrocytes, microglial cells, and infiltrating monocyte-derived macrophages play dual beneficial/harmful effects, via a panel of pro- or anti-inflammatory cytokines, chemokines, neurotrophic and neurogenic transcription factors. Notably, with age, microglia may adopt a potent neurotoxic, pro-inflammatory "primed" (M1) phenotype when challenged with inflammatory or neurotoxic stimuli that hamper brain's own restorative potential and inhibit endogenous neurorepair mechanisms. In the last decade we have provided evidence for a major role of microglial crosstalk with astrocytes, mDA neurons and neural stem progenitor cells (NSCs) in the MPTP- (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-) mouse model of PD, and identified Wnt/β-catenin signaling, a pivotal morphogen for mDA neurodevelopment, neuroprotection, and neuroinflammatory modulation, as a critical actor in glia-neuron and glia-NSCs crosstalk. With age however, Wnt signaling and glia-NSC-neuron crosstalk become dysfunctional with harmful consequences for mDA neuron plasticity and repair. These findings are of importance given the deregulation of Wnt signaling in PD and the emerging link between most PD related genes, Wnt signaling and inflammation. Especially, in light of the expanding field of microRNAs and inflammatory PD-related genes as modulators of microglial-proinflammatory status, uncovering the complex molecular circuitry linking PD and neuroinflammation will permit the identification of new druggable targets for the cure of the disease. Here we summarize recent findings unveiling major microglial inflammatory and oxidative stress pathways converging in the regulation of Wnt/β-catenin signaling, and reciprocally, the ability of Wnt signaling pathways to modulate microglial activation in PD. Unraveling the key factors and conditons promoting the switch of the proinflammatory M1 microglia status into a neuroprotective and regenerative M2 phenotype will have important consequences for neuroimmune interactions and neuronal outcome under inflammatory and/or neurodegenerative conditions.
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http://dx.doi.org/10.3389/fnagi.2018.00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816064PMC
February 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

Evaluation of RGD functionalization in hybrid hydrogels as 3D neural stem cell culture systems.

Biomater Sci 2018 Feb;6(3):501-510

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, via Mancinelli 7, 20131 Milan, Italy.

The use of neural stem cells (NSCs) in cell therapy has become a powerful tool used for the treatment of central nervous system diseases, including traumatic brain and spinal cord injuries. However, a significant drawback is related to the limited viability after transplantation in situ. The design of three-dimensional (3D) scaffolds that are capable of resembling the architecture and physico-chemical features of an extracellular environment could be a suitable approach to improve cell survival and preserve their cellular active phase over time. In this study, we investigated NSC adhesion and proliferation in hydrogel systems. In particular, we evaluated the effect of RGD binding domains on cell fate within the polymeric scaffold. The introduction of a tripeptide via hydrogel chemical functionalization improved the percentage of proliferating cells until 8 days after seeding when compared to the unmodified scaffold. The beneficial effects of this 3D culture system was further evident when compared to a NSC monolayer (2D) culture, resulting in an approximately 40% increase in cells in the active phases at 4 and 8 days, and maintained a difference of 25% until 21 days after seeding.
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http://dx.doi.org/10.1039/c7bm01056gDOI Listing
February 2018

Wnt3a promotes pro-angiogenic features in macrophages in vitro: Implications for stroke pathology.

Exp Biol Med (Maywood) 2018 01 4;243(1):22-28. Epub 2017 Dec 4.

1 Department of Clinical Neurosciences - Division of Stem Cell Neurobiology, Wellcome Trust-Medical Research Council Stem Cell Institute and NIHR Biomedical Research Centre, 151895 University of Cambridge , Cambridge CB2 0HA, UK.

Wnt3a is implicated in several key cellular processes and its expression has been reported in different cell types. Here, we report a novel function for Wnt3a in macrophages, whose exposure to this ligand shifts them towards a pro-angiogenic phenotype capable, under oxygen and glucose deprivation, of inducing in vitro tubular pattern structures in endothelial cells resembling capillary-like vasculature. These newly acquired angiogenetic features also include increased proliferation and migration and surprisingly, an increase in cell death. This work provides a new link between Wnt3a and macrophage-mediated angiogenesis under glucose and oxygen deprivation in vitro, which are worth further investigation in pathological conditions including stroke, where the stimulation of the angiogenic process might help to recovery after tissue injury Impact statement This work provides a new link between Wnt3a and macrophage-mediated angiogenesis under glucose and oxygen deprivation in vitro. Our results reveal how Wnt3a shifts macrophages towards a pro-angiogenic phenotype, which is able-in absence of both glucose and oxygen-of inducing angiogenesis in vitro, thus pointing to a synergy between the activation of the pathway and the hypoxia scenario. This work also demonstrates that modulation of cell death is key in order to explain the observed angiogenic effects. We consider all these findings of significant importance, since no connection between Wnt3a, macrophages, and angiogenesis has been established so far. Furthermore, we do believe that this work provides new and interesting results, with Wnt signaling pathway emerging as an interesting target mediating beneficial outcomes during the inflammatory response undoubtedly linked to stroke pathology, where angiogenesis has been already proposed as a potential mechanism to promote recovery after the injury.
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http://dx.doi.org/10.1177/1535370217746392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788164PMC
January 2018
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