[Show abstract][Hide abstract] ABSTRACT: a r t i c l e i n f o Hypothermia is potently neuroprotective but poor mechanistic understanding has restricted its clinical use. Rodent studies indicate that hypothermia can elicit preconditioning, wherein a subtoxic cellular stress confers resistance to an otherwise lethal injury. The molecular basis of this preconditioning remains obscure. Here we explore molecular effects of cooling using functional cortical neurons differentiated from human pluripotent stem cells (hCNs). Mild-to-moderate hypothermia (28–32 °C) induces cold-shock protein expression and mild endoplasmic reticulum (ER) stress in hCNs, with full activation of the unfolded protein response (UPR). Chemical block of a principal UPR pathway mitigates the protective effect of cooling against oxidative stress, whilst pre-cooling neurons abrogates the toxic injury produced by the ER stressor tunicamycin. Cold-stress thus preconditions neurons by upregulating adaptive chaperone-driven pathways of the UPR in a manner that precipitates ER-hormesis. Our findings establish a novel arm of neurocryobiology that could reveal multiple therapeutic targets for acute and chronic neuronal injury.
[Show abstract][Hide abstract] ABSTRACT: Coordinated development of brain stem and spinal target neurons is pivotal for the emergence of a precisely functioning locomotor system. Signals that match the development of these far-apart regions of the central nervous system may be redeployed during spinal cord regeneration. Here we show that descending dopaminergic projections from the brain promote motor neuron generation at the expense of V2 interneurons in the developing zebrafish spinal cord by activating the D4a receptor, which acts on the hedgehog pathway. Inhibiting this essential signal during early neurogenesis leads to a long-lasting reduction of motor neuron numbers and impaired motor responses of free-swimming larvae. Importantly, during successful spinal cord regeneration in adult zebrafish, endogenous dopamine promotes generation of spinal motor neurons, and dopamine agonists augment this process. Hence, we describe a supraspinal control mechanism for the development and regeneration of specific spinal cell types that uses dopamine as a signal.
[Show abstract][Hide abstract] ABSTRACT: Autoimmune voltage-gated potassium channelopathies represent a wide and expanding spectrum of neurological conditions. We present a case demonstrating the phenotypic heterogeneity of antivoltage-gated potassium channels (VGKC)-associated disorders. Such cases may easily be dismissed as functional disorders at first presentation. We propose that there must remain a high index of suspicion for antiVGKC-associated disorders in cases where there are transient neurological disturbances in atypical spatial and temporal distributions.
Case Reports 01/2013; 2013. DOI:10.1136/bcr-2012-007742
[Show abstract][Hide abstract] ABSTRACT: Multiple Sclerosis (MS) is an inflammatory demyelinating neurodegenerative disorder of the brain and spinal cord that causes significant disability in young adults. Although the precise aetiopathogenesis of MS remains unresolved, its pathological hallmarks include inflammation, demyelination, axonal injury (acute and chronic), astrogliosis and variable remyelination. Despite major recent advances in therapeutics for the early stage of the disease there are currently no disease modifying treatments for the progressive stage of disease, whose pathological substrate is axonal degeneration. This represents the great and unmet clinical need in MS. Against this background, human stem cells offer promise both to improve understanding of disease mechanism(s) through in-vitro modeling as well as potentially direct use to supplement and promote remyelination, an endogenous reparative process where entire myelin sheaths are restored to demyelinated axons. Conceptually, stem cells can act directly to myelinate axons or indirectly through different mechanisms to promote endogenous repair; importantly these two mechanisms of action are not mutually exclusive. We propose that discovery of novel methods to invoke or enhance remyelination in MS may be the most effective therapeutic strategy to limit axonal damage and instigate restoration of structure and function in this debilitating condition. Human stem cell derived neurons and glia, including patient specific cells derived through reprogramming, provide an unprecedented experimental system to model MS “in a dish” as well as enable high-throughput drug discovery. Finally, we speculate upon the potential role for stem cell based therapies in MS.
International Journal of Molecular Sciences 12/2012; 13(11):14470-91. DOI:10.3390/ijms131114470 · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A major goal in regenerative medicine is the predictable manipulation of human embryonic stem cells (hESCs) to defined cell fates that faithfully represent their somatic counterparts. Directed differentiation of hESCs into neuronal populations has galvanized much interest into their potential application in modelling neurodegenerative disease. However, neurodegenerative diseases are age-related, and therefore establishing the maturational comparability of hESC-derived neural derivatives is critical to generating accurate in vitro model systems. We address this issue by comparing genome-wide, exon-specific expression analyses of pluripotent hESCs, multipotent neural precursor cells and a terminally differentiated enriched neuronal population to expression data from post-mortem foetal and adult human brain samples. We show that hESC-derived neuronal cultures (using a midbrain differentiation protocol as a prototypic example of lineage restriction), while successful in generating physiologically functional neurons, are closer to foetal than adult human brain in terms of molecular maturation. These findings suggest that developmental stage has a more dominant influence on the cellular transcriptome than regional identity. In addition, we demonstrate that developmentally regulated gene splicing is common, and potentially a more sensitive measure of maturational state than gene expression profiling alone. In summary, this study highlights the value of genomic indices in refining and validating optimal cell populations appropriate for modelling ageing and neurodegeneration.
Journal of Neurochemistry 06/2012; 122(4):738-51. DOI:10.1111/j.1471-4159.2012.07825.x · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: More than half of patients with multiple sclerosis have progressive disease characterised by accumulating disability. The absence of treatments for progressive multiple sclerosis represents a major unmet clinical need. On the basis of evidence that mesenchymal stem cells have a beneficial effect in acute and chronic animal models of multiple sclerosis, we aimed to assess the safety and efficacy of these cells as a potential neuroprotective treatment for secondary progressive multiple sclerosis.
Patients with secondary progressive multiple sclerosis involving the visual pathways (expanded disability status score 5·5-6·5) were recruited from the East Anglia and north London regions of the UK. Participants received intravenous infusion of autologous bone-marrow-derived mesenchymal stem cells in this open-label study. Our primary objective was to assess feasibility and safety; we compared adverse events from up to 20 months before treatment until up to 10 months after the infusion. As a secondary objective, we chose efficacy outcomes to assess the anterior visual pathway as a model of wider disease. Masked endpoint analyses was used for electrophysiological and selected imaging outcomes. We used piecewise linear mixed models to assess the change in gradients over time at the point of intervention. This trial is registered with ClinicalTrials.gov, number NCT00395200.
We isolated, expanded, characterised, and administered mesenchymal stem cells in ten patients. The mean dose was 1·6×10(6) cells per kg bodyweight (range 1·1-2·0). One patient developed a transient rash shortly after treatment; two patients had self-limiting bacterial infections 3-4 weeks after treatment. We did not identify any serious adverse events. We noted improvement after treatment in visual acuity (difference in monthly rates of change -0·02 logMAR units, 95% CI -0·03 to -0·01; p=0·003) and visual evoked response latency (-1·33 ms, -2·44 to -0·21; p=0·020), with an increase in optic nerve area (difference in monthly rates of change 0·13 mm(2), 0·04 to 0·22; p=0·006). We did not identify any significant effects on colour vision, visual fields, macular volume, retinal nerve fibre layer thickness, or optic nerve magnetisation transfer ratio.
Autologous mesenchymal stem cells were safely given to patients with secondary progressive multiple sclerosis in our study. The evidence of structural, functional, and physiological improvement after treatment in some visual endpoints is suggestive of neuroprotection.
Medical Research Council, Multiple Sclerosis Society of Great Britain and Northern Ireland, Evelyn Trust, NHS National Institute for Health Research, Cambridge and UCLH Biomedical Research Centres, Wellcome Trust, Raymond and Beverly Sackler Foundation, and Sir David and Isobel Walker Trust.
The Lancet Neurology 02/2012; 11(2):150-6. DOI:10.1016/S1474-4422(11)70305-2 · 21.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Post-transcriptional regulation plays a major role in the generation of cell type diversity. In particular, alternative splicing increases diversification of transcriptome between tissues, in different cell types within a tissue, and even in different compartments of the same cell. The complexity of alternative splicing has increased during evolution. With increasing sophistication, however, comes greater potential for malfunction of these intricate processes. Indeed, recent years have uncovered a wealth of disease-causing mutations affecting RNA-binding proteins and non-coding regions on RNAs, highlighting the importance of studying disease mechanisms that act at the level of RNA processing. For instance, mutations in TARDBP and FUS, or a repeat expansion in the intronic region of the C9ORF72 gene, can all cause amyotrophic lateral sclerosis. We discuss how interspecies differences highlight the necessity for human model systems to complement existing non-human approaches to study neurodegenerative disorders. We conclude by discussing the improvements that could further increase the promise of human pluripotent stem for cell-based disease modeling. This article is part of a Special Issue entitled "RNA-Binding Proteins".
Brain research 01/2012; 1462:129-38. DOI:10.1016/j.brainres.2011.12.057 · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Motor neuron diseases (MND) such as amyotrophic lateral sclerosis and spinal muscular atrophy are devastating, progressive and ultimately fatal diseases for which there are no effective treatments. Recent evidence from systematic studies of animal models and human patients suggests that the neuromuscular junction (NMJ) is an important early target in MND, demonstrating functional and structural abnormalities in advance of pathological changes occurring in the motor neuron cell body. The ability to study pathological changes occurring at the NMJ in humans is therefore likely to be important for furthering our understanding of disease pathogenesis, and also for designing and testing new therapeutics. However, there are many practical and technical reasons why it is not possible to visualise or record from NMJs in pre- and early-symptomatic MND patients in vivo. Other approaches are therefore required. The development of stem cell technologies has opened up the possibility of creating human NMJs in vitro, using pluripotent cells generated from healthy individuals and patients with MND. This review covers historical attempts to develop mature and functional NMJs in vitro, using co-cultures of muscle and nerve from animals, and discusses how recent developments in the generation and specification of human induced pluripotent stem cells provides an opportunity to build on these previous successes to recapitulate human neuromuscular connectivity in vitro.
Journal of Anatomy 12/2011; 220(2):122-30. DOI:10.1111/j.1469-7580.2011.01459.x · 2.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The glial environment is an important determinant of neuronal health in experimental models of neurodegeneration. Specifically, astrocytes have been shown, dependent on context, to be both injurious and protective. Human pluripotent stem cells offer a powerful new system to improve our understanding of the mechanisms underlying astrocyte-mediated neuroprotection. Here, we describe a human embryonic stem cell (HESC)-based system to assess the scope and mechanism of human astrocyte-mediated neuroprotection. We first report the generation of enriched and functional HESC-derived astrocytes, by combining BMP-mediated Smad and LIF-mediated JAK-STAT signalling. These astrocytes promote the protection of HESC-derived neurons against oxidative insults. Moreover, their neuroprotective capacity can be greatly enhanced by treatment with the nuclear factor-erythroid 2-related factor 2 (Nrf2)-activating triterpenoid 1[2-Cyano-3,12-dioxool-eana-1,9(11)-dien-28-oyl] trifluoroethylamide (CDDO(TFEA)). Activation of the transcription factor Nrf2 in human astrocytes by CDDO(TFEA) treatment induced expression of the glutamate-cysteine ligase (GCL) catalytic subunit, leading to enhanced GCL activity and glutathione production, and strong neuroprotection against H(2)O(2). This enhanced neuroprotection was found to be dependent on astrocytic GCL activity, unlike the basal neuroprotection afforded by untreated astrocytes. Direct treatment of HESC-derived neurons with CDDO(TFEA) elicited no induction of Nrf2 target genes, nor any neuroprotection. Thus, human astrocytes can mediate neuroprotection through glutathione-dependent and glutathione-independent mechanisms, and represent a therapeutic target for human disorders associated with neuronal oxidative stress.
Cell death and differentiation 11/2011; 19(5):779-87. DOI:10.1038/cdd.2011.154 · 8.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chondroitin sulfate proteoglycans (CSPGs) are upregulated after CNS lesions, where they inhibit axon regeneration. In order for axon growth and regeneration to occur, surface integrin receptors must interact with surrounding extracellular matrix molecules. We have explored the hypothesis that CSPGs inhibit regeneration by inactivating integrins and that forcing integrins into an active state might overcome this inhibition. Using cultured rat sensory neurons, we show that the CSPG aggrecan inhibits laminin-mediated axon growth by impairing integrin signaling via decreasing phosphorylated FAK (pFAK) and pSrc levels, without affecting surface integrin levels. Forcing integrin activation and signaling by manganese or an activating antibody TS2/16 reversed the inhibitory effect of aggrecan on mixed aggrecan/laminin surfaces, and enhanced axon growth from cultured rat sensory neurons (manganese) and human embryonic stem cell-derived motoneurons (TS2/16). The inhibitory effect of Nogo-A can also be reversed by integrin activation. These results suggest that inhibition by CSPGs can act via inactivation of integrins, and that activation of integrins is a potential method for improving axon regeneration after injury.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 04/2011; 31(17):6289-95. DOI:10.1523/JNEUROSCI.0008-11.2011 · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: No treatments are currently available that slow, stop, or reverse disease progression in established multiple sclerosis (MS). The Mesenchymal Stem Cells in Multiple Sclerosis (MSCIMS) trial tests the safety and feasibility of treatment with a candidate cell-based therapy, and will inform the wider challenge of designing early phase clinical trials to evaluate putative neuroprotective therapies in progressive MS. Illustrated by the MSCIMS trial protocol, we describe a novel methodology based on detailed assessment of the anterior visual pathway as a model of wider disease processes--the "sentinel lesion approach".
MSCIMS is a phase IIA study of autologous mesenchymal stem cells (MSCs) in secondary progressive MS. A pre-test : post-test design is used with healthy controls providing normative data for inter-session variability. Complementary eligibility criteria and outcomes are used to select participants with disease affecting the anterior visual pathway.
Ten participants with MS and eight healthy controls were recruited between October 2008 and March 2009. Mesenchymal stem cells were successfully isolated, expanded and characterised in vitro for all participants in the treatment arm.
In addition to determining the safety and feasibility of the intervention and informing design of future studies to address efficacy, MSCIMS adopts a novel strategy for testing neuroprotective agents in MS--the sentinel lesion approach--serving as proof of principle for its future wider applicability.
[Show abstract][Hide abstract] ABSTRACT: A major challenge in neurobiology is to understand mechanisms underlying human neuronal diversification. Motor neurons (MNs) represent a diverse collection of neuronal subtypes, displaying differential vulnerability in different human neurodegenerative diseases. The ability to manipulate cell subtype diversification is critical to establish accurate, clinically relevant in vitro disease models. Retinoid signalling contributes to caudal precursor specification and subsequent MN subtype diversification. Here we investigate the necessity for retinoic acid in motor neurogenesis from human embryonic stem cells. We show that activin/nodal signalling inhibition, followed by sonic hedgehog agonist treatment, is sufficient for MN precursor specification, which occurs even in the presence of retinoid pathway antagonists. Importantly, precursors mature into HB9/ChAT-expressing functional MNs. Furthermore, retinoid-independent motor neurogenesis results in a ground state biased to caudal, medial motor columnar identities from which a greater retinoid-dependent diversity of MNs, including those of lateral motor columns, can be selectively derived in vitro.
[Show abstract][Hide abstract] ABSTRACT: Public and media interest in the potential applications of stem cells in regenerative neurology has led to growing hope and expectation. This interest is heightened by the current paucity of treatments available for neurodegenerative diseases and their generally poor prognosis. Patient discussions about stem cells are therefore a common occurrence in clinical practice, requiring neurologists to offer clear and accurate information. In the context of a complex and rapidly evolving field, this can be extremely challenging. Here we address issues around stem cell populations relevant to regenerative neurology, including the opportunities they offer for research and their potential application as direct therapies, concluding with a pragmatic assessment of the likely clinical benefits of stem cell research.
[Show abstract][Hide abstract] ABSTRACT: TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.
[Show abstract][Hide abstract] ABSTRACT: Intracellular filamentous deposits containing microtubule-associated protein tau constitute a defining characteristic of many neurodegenerative disorders. Current experimental models to study tau pathology in vitro do not usually recapitulate the tau expression pattern characteristic of adult human brain. In this study, we have investigated whether human embryonic stem cell-derived neurons could be a good model to study human tau distribution, function and dysfunction.
Using RT-PCR, immunohistochemistry, western blotting and cell transfections we have investigated whether all 6 adult human brain tau isoforms are expressed in neurons derived from human embryonic and fetal stem cells and whether 4 repeat tau over-expression alone, or with the F3 tau repeat fragment, (amino acid 258-380 of the 2N4R tau isoform with the ΔK280 mutation) affects tau distribution. We found that the shortest 3 repeat tau isoform, similarly to human brain, is the first to be expressed during neuronal differentiation while the other 5 tau isoforms are expressed later. Over expression of tau with 4 repeats affects tau cellular distribution and the short tau F3 fragment appears to increase tau phosphorylation but this effect does not appear to be toxic for the cell.
Our results indicate that human embryonic stem cell-derived neurons express all 6 tau isoforms and are a good model in which to study tau physiology and pathology.
PLoS ONE 11/2010; 5(11):e13947. DOI:10.1371/journal.pone.0013947 · 3.53 Impact Factor