Publications by authors named "Bilada Bilican"

23 Publications

  • Page 1 of 1

CellSIUS provides sensitive and specific detection of rare cell populations from complex single-cell RNA-seq data.

Genome Biol 2019 07 17;20(1):142. Epub 2019 Jul 17.

Novartis Institutes for Biomedical Research, Basel, Switzerland.

We develop CellSIUS (Cell Subtype Identification from Upregulated gene Sets) to fill a methodology gap for rare cell population identification for scRNA-seq data. CellSIUS outperforms existing algorithms for specificity and selectivity for rare cell types and their transcriptomic signature identification in synthetic and complex biological data. Characterization of a human pluripotent cell differentiation protocol recapitulating deep-layer corticogenesis using CellSIUS reveals unrecognized complexity in human stem cell-derived cellular populations. CellSIUS enables identification of novel rare cell populations and their signature genes providing the means to study those populations in vitro in light of their role in health and disease.
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http://dx.doi.org/10.1186/s13059-019-1739-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637521PMC
July 2019

Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection.

Cell Stem Cell 2016 12;19(6):703-708

Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Zika virus (ZIKV) can cross the placental barrier, resulting in infection of the fetal brain and neurological defects including microcephaly. The cellular tropism of ZIKV and the identity of attachment factors used by the virus to gain access to key cell types involved in pathogenesis are under intense investigation. Initial studies suggested that ZIKV preferentially targets neural progenitor cells (NPCs), providing an explanation for the developmental phenotypes observed in some pregnancies. The AXL protein has been nominated as a key attachment factor for ZIKV in several cell types including NPCs. However, here we show that genetic ablation of AXL has no effect on ZIKV entry or ZIKV-mediated cell death in human induced pluripotent stem cell (iPSC)-derived NPCs or cerebral organoids. These findings call into question the utility of AXL inhibitors for preventing birth defects after infection and suggest that further studies of viral attachment factors in NPCs are needed.
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http://dx.doi.org/10.1016/j.stem.2016.11.011DOI Listing
December 2016

Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons.

Elife 2016 10 1;5. Epub 2016 Oct 1.

School of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom.

Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents.
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http://dx.doi.org/10.7554/eLife.20337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092045PMC
October 2016

Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2.

Nat Commun 2015 May 13;6:7066. Epub 2015 May 13.

Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, UK.

Forebrain neurons have weak intrinsic antioxidant defences compared with astrocytes, but the molecular basis and purpose of this is poorly understood. We show that early in mouse cortical neuronal development in vitro and in vivo, expression of the master-regulator of antioxidant genes, transcription factor NF-E2-related-factor-2 (Nrf2), is repressed by epigenetic inactivation of its promoter. Consequently, in contrast to astrocytes or young neurons, maturing neurons possess negligible Nrf2-dependent antioxidant defences, and exhibit no transcriptional responses to Nrf2 activators, or to ablation of Nrf2's inhibitor Keap1. Neuronal Nrf2 inactivation seems to be required for proper development: in maturing neurons, ectopic Nrf2 expression inhibits neurite outgrowth and aborization, and electrophysiological maturation, including synaptogenesis. These defects arise because Nrf2 activity buffers neuronal redox status, inhibiting maturation processes dependent on redox-sensitive JNK and Wnt pathways. Thus, developmental epigenetic Nrf2 repression weakens neuronal antioxidant defences but is necessary to create an environment that supports neuronal development.
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http://dx.doi.org/10.1038/ncomms8066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4441249PMC
May 2015

Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.

J Physiol 2014 Oct 28;592(19):4353-63. Epub 2014 Aug 28.

Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Patrick Wild Centre, University of Edinburgh, Edinburgh, EH8 9XD, UK

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition - a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits.
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http://dx.doi.org/10.1113/jphysiol.2014.278994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215781PMC
October 2014

Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models.

Nat Chem Biol 2014 Aug 29;10(8):677-85. Epub 2014 Jun 29.

1] Gladstone Institute of Neurologic Disease, San Francisco, California, USA. [2] Department of Neurology, University of California-San Francisco Medical Center, San Francisco, California, USA. [3] Biomedical Sciences Graduate Program, University of California-San Francisco, San Francisco, California, USA. [4] Keck Program in Brain Cell Engineering, Gladstone Institutes, San Francisco, California, USA. [5] Department of Physiology, University of California-San Francisco, San Francisco, California, USA. [6] Taube-Koret Center for Neurodegenerative Disease Research, San Francisco, California, USA. [7] Hellman Family Foundation Alzheimer's Disease Research Program, San Francisco, California, USA. [8] Roddenberry Stem Cell Program, San Francisco, California, USA.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology--cytoplasmic inclusions rich in transactive response element DNA-binding protein of 43 kDa (TDP43). Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we show that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity and discovered that pathogenic mutations shorten TDP43 half-life. New compounds that stimulate autophagy improved TDP43 clearance and localization and enhanced survival in primary murine neurons and in human stem cell-derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance.
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http://dx.doi.org/10.1038/nchembio.1563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106236PMC
August 2014

Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells.

PLoS One 2014 20;9(3):e91269. Epub 2014 Mar 20.

Department of Clinical Neuroscience, King's College London, London, United Kingdom.

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain, including the variant M337V, which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool, we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions, which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA, cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells, thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091269PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961241PMC
December 2014

Maturation of AMPAR composition and the GABAAR reversal potential in hPSC-derived cortical neurons.

J Neurosci 2014 Mar;34(11):4070-5

Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; and Euan MacDonald Centre for MND Research, Centre for Clinical Brain Sciences, and MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom.

Rodent-based studies have shown that neurons undergo major developmental changes to ion channel expression and ionic gradients that determine their excitation-inhibition balance. Neurons derived from human pluripotent stem cells theoretically offer the potential to study classical developmental processes in a human-relevant system, although this is currently not well explored. Here, we show that excitatory cortical-patterned neurons derived from multiple human pluripotent stem cell lines exhibit native-like maturation changes in AMPAR composition such that there is an increase in the expression of GluA2(R) subunits. Moreover, we observe a dynamic shift in intracellular Cl- levels, which determines the reversal potential of GABAAR-mediated currents and is influenced by neurotrophic factors. The shift is concomitant with changes in KCC2 and NKCC1 expression. Because some human diseases are thought to involve perturbations to AMPAR GluA2 content and others in the chloride reversal potential, human stem-cell-derived neurons represent a valuable tool for studying these fundamental properties.
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http://dx.doi.org/10.1523/JNEUROSCI.5410-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951701PMC
March 2014

Axonal transport of TDP-43 mRNA granules is impaired by ALS-causing mutations.

Neuron 2014 Feb;81(3):536-543

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

The RNA-binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP-43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43.
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http://dx.doi.org/10.1016/j.neuron.2013.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939050PMC
February 2014

Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells.

PLoS One 2014 17;9(1):e85932. Epub 2014 Jan 17.

Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom ; MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom ; Centre for Neuroregeneration, University of Edinburgh, Edinburgh, United Kingdom.

Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O2. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O2 (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085932PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895023PMC
September 2014

High yields of oligodendrocyte lineage cells from human embryonic stem cells at physiological oxygen tensions for evaluation of translational biology.

Stem Cell Reports 2013 31;1(5):437-50. Epub 2013 Oct 31.

Department of Clinical Neurosciences, Cambridge University, Cambridge CB2 0PY, UK ; Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute and Department of Veterinary Medicine, Cambridge University, Cambridge CB3 0ES, UK.

We have established and efficient system to specify NG2/PDGF-Rα/OLIG2+ oligodendrocyte precursor cells (OPCs) from human embryonic stem cells (hESCs) at low, physiological (3%) oxygen levels. This was achieved via both forebrain and spinal cord origins, with up to 98% of cells expressing NG2. Developmental insights reveal a critical role for fibroblast growth factor 2 (FGF-2) in OLIG2 induction via ventral forebrain pathways. The OPCs mature in vitro to express O4 (46%) and subsequently become galactocerebroside (GALC), O1, and myelin basic protein-positive (MBP+) multibranching oligodendrocytes. These were cultured alongside hESC-derived neurons. The electrophysiological properties of human OPCs are similar to those of rat OPCs, with large voltage-gated sodium currents and the ability to fire action potentials. Exposure to a selective retinoid X receptor agonist increased the proportion of O4+ oligodendrocytes that express MBP from 5% to 30%. Thus, we have established a developmentally engineered system to investigate the biological properties of human OPCs and test the effects of putative remyelinating agents prior to clinical application.
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http://dx.doi.org/10.1016/j.stemcr.2013.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841262PMC
October 2015

Unpicking neurodegeneration in a dish with human pluripotent stem cells: one cell type at a time.

Cell Cycle 2013 Aug 11;12(15):2339-40. Epub 2013 Jul 11.

Euan MacDonald Centre for Motor Neurone Disease Research; Centre for Neuroregeneration and Medical Research Council Centre for Regenerative Medicine; University of Edinburgh, Edinburgh, UK.

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http://dx.doi.org/10.4161/cc.25705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841307PMC
August 2013

Comment on "Drug screening for ALS using patient-specific induced pluripotent stem cells".

Sci Transl Med 2013 Jun;5(188):188le2

Egawa et al. recently showed the value of patient-specific induced pluripotent stem cells (iPSCs) for modeling amyotrophic lateral sclerosis in vitro. Their study and our work highlight the need for complementary assays to detect small, but potentially important, phenotypic differences between control iPSC lines and those carrying disease mutations.
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http://dx.doi.org/10.1126/scitranslmed.3005065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936961PMC
June 2013

Neural precursor cells cultured at physiologically relevant oxygen tensions have a survival advantage following transplantation.

Stem Cells Transl Med 2013 Jun 15;2(6):464-72. Epub 2013 May 15.

Department of Clinical Neurosciences, University Medical Center, the Netherlands.

Traditionally, in vitro stem cell systems have used oxygen tensions that are far removed from the in vivo situation. This is particularly true for the central nervous system, where oxygen (O2) levels range from 8% at the pia to 0.5% in the midbrain, whereas cells are usually cultured in a 20% O2 environment. Cell transplantation strategies therefore typically introduce a stress challenge at the time of transplantation as the cells are switched from 20% to 3% O2 (the average in adult organs). We have modeled the oxygen stress that occurs during transplantation, demonstrating that in vitro transfer of neonatal rat cortical neural precursor cells (NPCs) from a 20% to a 3% O2 environment results in significant cell death, whereas maintenance at 3% O2 is protective. This survival benefit translates to the in vivo environment, where culture of NPCs at 3% rather than 20% O2 approximately doubles survival in the immediate post-transplantation phase. Furthermore, NPC fate is affected by culture at low, physiological O2 tensions (3%), with particularly marked effects on the oligodendrocyte lineage, both in vitro and in vivo. We propose that careful consideration of physiological oxygen environments, and particularly changes in oxygen tension, has relevance for the practical approaches to cellular therapies.
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http://dx.doi.org/10.5966/sctm.2012-0144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3673758PMC
June 2013

Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy.

Proc Natl Acad Sci U S A 2013 Mar 11;110(12):4697-702. Epub 2013 Feb 11.

Euan MacDonald Centre for Motor Neurone Disease Research, Centre for Neuroregeneration, and Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, United Kingdom.

Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.
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http://dx.doi.org/10.1073/pnas.1300398110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3607024PMC
March 2013

Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability.

Proc Natl Acad Sci U S A 2012 Apr 26;109(15):5803-8. Epub 2012 Mar 26.

Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4SB, United Kingdom.

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening.
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http://dx.doi.org/10.1073/pnas.1202922109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326463PMC
April 2012

Cell-mediated neuroprotection in a mouse model of human tauopathy.

J Neurosci 2010 Jul;30(30):9973-83

Euan MacDonald Centre for Motor Neurone Disease Research, Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom.

Tau protein in a hyperphosphorylated state makes up the intracellular inclusions of several neurodegenerative diseases, including Alzheimer's disease and cases of frontotemporal dementia. Mutations in Tau cause familial forms of frontotemporal dementia, establishing that dysfunction of tau protein is sufficient to cause neurodegeneration and dementia. Transgenic mice expressing human mutant tau in neurons exhibit the essential features of tauopathies, including neurodegeneration and abundant filaments composed of hyperphosphorylated tau. Here we show that a previously described mouse line transgenic for human P301S tau exhibits an age-related, layer-specific loss of superficial cortical neurons, similar to what has been observed in human frontotemporal dementias. We also show that focal neural precursor cell implantation, resulting in glial cell differentiation, leads to the sustained rescue of cortical neurons. Together with evidence indicating that astrocyte transplantation may be neuroprotective, our findings suggest a beneficial role for glial cell-based repair in neurodegenerative diseases.
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http://dx.doi.org/10.1523/JNEUROSCI.0834-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633376PMC
July 2010

Signaling pathways controlling pluripotency and early cell fate decisions of human induced pluripotent stem cells.

Stem Cells 2009 Nov;27(11):2655-66

Laboratory for Regenerative Medicine, University of Cambridge, Cambridge CB2 0SZ, United Kingdom.

Human pluripotent stem cells from embryonic origins and those generated from reprogrammed somatic cells share many characteristics, including indefinite proliferation and a sustained capacity to differentiate into a wide variety of cell types. However, it remains to be demonstrated whether both cell types rely on similar mechanisms to maintain their pluripotent status and to control their differentiation. Any differences in such mechanisms would suggest that reprogramming of fibroblasts to generate induced pluripotent stem cells (iPSCs) results in novel states of pluripotency. In that event, current methods for expanding and differentiating human embryonic stem cells (ESCs) might not be directly applicable to human iPSCs. However, we show here that human iPSCs rely on activin/nodal signaling to control Nanog expression and thereby maintain pluripotency, thus revealing their mechanistic similarity to human ESCs. We also show that growth factors necessary and sufficient for achieving specification of human ESCs into extraembryonic tissues, neuroectoderm, and mesendoderm also drive differentiation of human iPSCs into the same tissues. Importantly, these experiments were performed in fully chemically defined medium devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Together these data reveal that human iPSCs rely on mechanisms similar to human ESCs to maintain their pluripotency and to control their differentiation, showing that these pluripotent cell types are functionally equivalent.
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http://dx.doi.org/10.1002/stem.199DOI Listing
November 2009

Induction of Olig2 precursors by FGF involves BMP signalling blockade at the Smad level.

PLoS One 2008 Aug 6;3(8):e2863. Epub 2008 Aug 6.

Department of Clinical Neurosciences and Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom.

During normal development oligodendrocyte precursors (OPCs) are generated in the ventral spinal cord in response to Sonic hedgehog (Shh) signalling. There is also a second, late wave of oligodendrogenesis in the dorsal spinal cord independent of Shh activity. Two signalling pathways, controlled by bone morphogenetic protein and fibroblast growth factor (FGF), are active players in dorsal spinal cord specification. In particular, BMP signalling from the roof plate has a crucial role in setting up dorsal neural identity and its inhibition is sufficient to generate OPCs both in vitro and in vivo. In contrast, FGF signalling can induce OPC production from dorsal spinal cord cultures in vitro. In this study, we examined the cross-talk between mitogen-activated protein kinase (MAPK) and BMP signalling in embryonic dorsal spinal cord cultures at the SMAD1/5/8 (SMAD1) transcription factor level, the main effectors of BMP activity. We have previously shown that FGF2 treatment of neural precursor cells (NPCs) derived from rat E14 dorsal spinal cord is sufficient to generate OPCs in vitro. Utilising the same system, we now show that FGF prevents BMP-induced nuclear localisation of SMAD1-phosphorylated at the C-terminus (C-term-pSMAD1). This nuclear exclusion of C-term-pSMAD1 is dependent on MAPK activity and correlates with OLIG2 upregulation, the obligate transcription factor for oligodendrogenesis. Furthermore, inhibition of the MAPK pathway abolishes OLIG2 expression. We also show that SMAD4, which acts as a common partner for receptor-regulated Smads including SMAD1, associates with a Smad binding site in the Olig2 promoter and dissociates from it upon differentiation. Taken together, these results suggest that FGF can promote OPC generation from embryonic NPCs by counteracting BMP signalling at the Smad1 transcription factor level and that Smad-containing transcriptional complexes may be involved in direct regulation of the Olig2 promoter.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002863PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483937PMC
August 2008

T-box factors: targeting to chromatin and interaction with the histone H3 N-terminal tail.

Pigment Cell Res 2007 Aug;20(4):279-87

Signalling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK.

T-box transcription factors play a crucial role in development where they are implicated in patterning and cell fate decisions. Tbx2 and Tbx3 have also been implicated in several cancers including melanoma, and can act as antisenescence factors through their ability to repress p19(ARF) and p21(CIP1) expression. Although several target genes for T-box factors have been identified, it is unknown whether this family of proteins can bind chromatin, a property that would facilitate the epigenetic reprogramming that occurs in both development and cancer progression. Here, we show that Tbx2 has the potential to recognize mitotic chromatin in a DNA-dependent fashion, can interact specifically with the histone H3 N-terminal tail, a property shared with Tbx4, Tbx5 and Tbx6, and can also recognize nucleosomal DNA, with binding to nucleosomes being antagonized by the presence of the histone tails. Strikingly, in vivo Tbx2 co-localization with pericentric heterochromatin appears to be regulated and ectopic expression of Tbx2 leads to severe mitotic defects. Taken together our results suggest that Tbx2, and most likely other members of the T-box family, are able to target chromatin and may indicate a role for the T-box factors in epigenetic reprogramming events.
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http://dx.doi.org/10.1111/j.1600-0749.2007.00389.xDOI Listing
August 2007

Cell cycle regulation of the T-box transcription factor tbx2.

Exp Cell Res 2006 Jul 6;312(12):2358-66. Epub 2006 May 6.

Signalling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK.

T-box transcription factors play key roles in development and in particular the determination or maintenance of cell fate. Tbx2 is a transcriptional repressor implicated in several developmental processes and which has also been implicated in cancer through its ability to suppress senescence via repression of the p19(ARF) and p21(Cip1) (CDKN1A) promoters. However, despite its importance, little is known about how Tbx2 may be regulated. Here, we show that Tbx2 protein expression is tightly regulated during cell cycle progression, with levels being low in G1, increasing in mid-S-phase and persisting at high levels though G2 until finally undergoing a dramatic reduction at the onset of mitosis. Moreover, in S-phase, Tbx2 is present at a subset of late, but not early, replication foci and a significant fraction of Tbx2 is tightly associated with the nucleus in small DNA-associated foci that do not correspond with telomeres, PML or cajal bodies. The results are consistent with Tbx2 playing a role in cell cycle progression and organization of subnuclear compartments.
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http://dx.doi.org/10.1016/j.yexcr.2006.03.033DOI Listing
July 2006

Saccharomyces cerevisiae C1D is implicated in both non-homologous DNA end joining and homologous recombination.

Mol Microbiol 2002 Nov;46(4):947-57

Bikent University, Molecular Biology and Genetics Department, Ankara, Turkey.

C1D is a gamma-irradiation inducible nuclear matrix protein that interacts with and activates the DNA-dependent protein kinase (DNA-PK) that is essential for the repair of the DNA double-strand breaks and V(D)J recombination. Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break-point junctions, and that over expression of C1D can induce p53-dependent apoptosis. Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination. To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue. We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non-homologous DNA end joining (NHEJ) and accurate DNA repair. In addition, using a novel plasmid-based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination. These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double-strand breaks.
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http://dx.doi.org/10.1046/j.1365-2958.2002.03224.xDOI Listing
November 2002

DNA damage-dependent interaction of the nuclear matrix protein C1D with Translin-associated factor X (TRAX).

J Cell Sci 2002 Jan;115(Pt 1):207-16

Bilkent University, Molecular Biology and Genetics Department, 06533, Bilkent, Ankara, Turkey.

The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination. C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon gamma-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo. In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX. Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus. We show that C1D and TRAX interact specifically in both yeast and mammalian cells. Interestingly, however, interaction of these two proteins in mammalian cells only occur following gamma-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX. Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization. These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation.
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January 2002