Publications by authors named "Alexis-Pierre Bemelmans"

48 Publications

Astrocytes close the mouse critical period for visual plasticity.

Science 2021 07;373(6550):77-81

Center for Interdisciplinary Research in Biology, Collège de France, CNRS UMR 7241, INSERM U1050, Labex Memolife, PSL Research University, Paris, France.

Brain postnatal development is characterized by critical periods of experience-dependent remodeling of neuronal circuits. Failure to end these periods results in neurodevelopmental disorders. The cellular processes defining critical-period timing remain unclear. Here, we show that in the mouse visual cortex, astrocytes control critical-period closure. We uncover the underlying pathway, which involves astrocytic regulation of the extracellular matrix, allowing interneuron maturation. Unconventional astrocyte connexin signaling hinders expression of extracellular matrix-degrading enzyme matrix metalloproteinase 9 (MMP9) through RhoA-guanosine triphosphatase activation. Thus, astrocytes not only influence the activity of single synapses but also are key elements in the experience-dependent wiring of brain circuits.
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http://dx.doi.org/10.1126/science.abf5273DOI Listing
July 2021

The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo.

Int J Mol Sci 2021 Jun 23;22(13). Epub 2021 Jun 23.

Université Paris-Saclay, CEA, CNRS, Laboratoire des Maladies Neurodégénératives, MIRCen, F-92265 Fontenay-aux-Roses, France.

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson's disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) "cell-autonomous". Here, we explored whether ΔLRRK2 could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2 or its "dead" kinase form, ΔLRRK2, and human α-syn with the A53T mutation (AAV-α-syn). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-syn alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-syn with AAV-ΔLRRK2 induced DA neuron degeneration that was significantly higher than that induced by AAV-α-syn alone or with AAV-ΔLRRK2. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2 alone through cell-autonomous mechanisms.
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http://dx.doi.org/10.3390/ijms22136760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268201PMC
June 2021

THY-Tau22 mouse model accumulates more tauopathy at late stage of the disease in response to microglia deactivation through TREM2 deficiency.

Neurobiol Dis 2021 Jul 18;155:105398. Epub 2021 May 18.

Université Paris-Saclay, CEA, CNRS, MIRCen, Laboratoire des Maladies Neurodégénératives, Fontenay-aux-Roses, France. Electronic address:

The role played by microglia has taken the center of the stage in the etiology of Alzheimer's disease (AD). Several genome-wide association studies carried out on large cohorts of patients have indeed revealed a large number of genetic susceptibility factors corresponding to genes involved in neuroinflammation and expressed specifically by microglia in the brain. Among these genes TREM2, a cell surface receptor expressed by microglia, arouses strong interest because its R47H variant confers a risk of developing AD comparable to the ε4 allele of the APOE gene. Since this discovery, a growing number of studies have therefore examined the role played by TREM2 in the evolution of amyloid plaques and neurofibrillary tangles, the two brain lesions characteristic of AD. Many studies report conflicting results, reflecting the complex nature of microglial activation in AD. Here, we investigated the impact of TREM2 deficiency in the THY-Tau22 transgenic line, a well-characterized model of tauopathy. Our study reports an increase in the severity of tauopathy lesions in mice deficient in TREM2 occurring at an advanced stage of the pathology. This exacerbation of pathology was associated with a reduction in microglial activation indicated by typical morphological features and altered expression of specific markers. However, it was not accompanied by any further changes in memory performance. Our longitudinal study confirms that a defect in microglial TREM2 signaling leads to an increase in neuronal tauopathy occurring only at late stages of the disease.
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http://dx.doi.org/10.1016/j.nbd.2021.105398DOI Listing
July 2021

Neuronal tau species transfer to astrocytes and induce their loss according to tau aggregation state.

Brain 2021 May;144(4):1167-1182

Université Paris-Saclay, CEA, CNRS, MIRCen, Laboratoire des Maladies Neurodégénératives, 92265, Fontenay-aux-Roses, France.

Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-pathological features of tauopathies. Although tau protein is highly enriched in neurons and poorly expressed by astrocytes, the origin of astrocytic tau is still elusive. Here, we used innovative gene transfer tools to model tauopathies in adult mouse brains and to investigate the origin of astrocytic tau. We showed in our adeno-associated virus (AAV)-based models and in Thy-Tau22 transgenic mice that astrocytic tau pathology can emerge secondarily to neuronal pathology. By designing an in vivo reporter system, we further demonstrated bidirectional exchanges of tau species between neurons and astrocytes. We then determined the consequences of tau accumulation in astrocytes on their survival in models displaying various status of tau aggregation. Using stereological counting of astrocytes, we report that, as for neurons, soluble tau species are highly toxic to some subpopulations of astrocytes in the hippocampus, whereas the accumulation of tau aggregates does not affect their survival. Thus, astrocytes are not mere bystanders of neuronal pathology. Our results strongly suggest that tau pathology in astrocytes may significantly contribute to clinical symptoms.
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http://dx.doi.org/10.1093/brain/awab011DOI Listing
May 2021

Local Translation in Perisynaptic Astrocytic Processes Is Specific and Changes after Fear Conditioning.

Cell Rep 2020 08;32(8):108076

Physiology and Physiopathology of the Gliovascular Unit Research Group, Center for Interdisciplinary Research in Biology (CIRB), Collège de France, CNRS Unité Mixte de Recherche 724, INSERM Unité 1050, Labex Memolife, PSL Research University, Paris, France. Electronic address:

Local translation is a conserved mechanism conferring cells the ability to quickly respond to local stimuli. In the brain, it has been recently reported in astrocytes, whose fine processes contact blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown. We study hippocampal perisynaptic astrocytic processes (PAPs) and show that they contain the machinery for translation. Using a refined immunoprecipitation technique, we characterize the entire pool of ribosome-bound mRNAs in PAPs and compare it with the one expressed in the whole astrocyte. We find that a specific pool of mRNAs is highly polarized at the synaptic interface. These transcripts encode an unexpected molecular repertoire, composed of proteins involved in iron homeostasis, translation, cell cycle, and cytoskeleton. Remarkably, we observe alterations in global RNA distribution and ribosome-bound status of some PAP-enriched transcripts after fear conditioning, indicating the role of astrocytic local translation in memory and learning.
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http://dx.doi.org/10.1016/j.celrep.2020.108076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450274PMC
August 2020

Complex roles for reactive astrocytes in the triple transgenic mouse model of Alzheimer disease.

Neurobiol Aging 2020 06 18;90:135-146. Epub 2020 Feb 18.

Université Paris-Saclay, CEA, CNRS, MIRCen, Laboratoire des Maladies Neurodégénératives, Fontenay-aux-Roses, France. Electronic address:

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we developed viral vectors targeting astrocytes that either activate or inhibit the Janus kinase-signal transducer and activator of transcription 3 (JAK2-STAT3) pathway, a central cascade controlling astrocyte reaction. We aimed to evaluate whether reactive astrocytes contribute to tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops tau hyper-phosphorylation and amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in 25% of the hippocampus of 3xTg-AD mice did not significantly influence tau phosphorylation or amyloid processing and deposition at early, advanced, and terminal disease stage. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve spatial memory in the Y maze but it did reduce anxiety in the elevated plus maze. Our unique approach to specifically manipulate reactive astrocytes in situ show they may impact behavioral outcomes without influencing tau or amyloid pathology.
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http://dx.doi.org/10.1016/j.neurobiolaging.2020.02.010DOI Listing
June 2020

Impairment of Glycolysis-Derived l-Serine Production in Astrocytes Contributes to Cognitive Deficits in Alzheimer's Disease.

Cell Metab 2020 03;31(3):503-517.e8

Université Paris-Saclay, CEA, CNRS, MIRCen, Laboratoire des Maladies Neurodégénératives, Fontenay-aux-Roses, France.

Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD.
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http://dx.doi.org/10.1016/j.cmet.2020.02.004DOI Listing
March 2020

rAAV-Mediated Cochlear Gene Therapy: Prospects and Challenges for Clinical Application.

J Clin Med 2020 Feb 21;9(2). Epub 2020 Feb 21.

INSERM - UMR 1051, Institut des Neurosciences de Montpellier, Hôpital Saint Eloi - Bâtiment INM, 80, rue Augustin Fliche - BP 74103, 34091 Montpellier, France.

Over the last decade, pioneering molecular gene therapy for inner-ear disorders have achieved experimental hearing improvements after a single local or systemic injection of adeno-associated, virus-derived vectors (rAAV for recombinant AAV) encoding an extra copy of a normal gene, or ribozymes used to modify a genome. These results hold promise for treating congenital or later-onset hearing loss resulting from monogenic disorders with gene therapy approaches in patients. In this review, we summarize the current state of rAAV-mediated inner-ear gene therapies including the choice of vectors and delivery routes, and discuss the prospects and obstacles for the future development of efficient clinical rAAV-mediated cochlear gene medicine therapy.
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http://dx.doi.org/10.3390/jcm9020589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073754PMC
February 2020

The intellectual disability protein Oligophrenin-1 controls astrocyte morphology and migration.

Glia 2020 09 19;68(9):1729-1742. Epub 2020 Feb 19.

Institut Cochin, INSERM UMR 1016, CNRS UMR 8104, Université de Paris, Paris, France.

Astrocytes are involved in several aspects of neuronal development and properties which are altered in intellectual disability (ID). Oligophrenin-1 is a RhoGAP protein implicated in actin cytoskeleton regulation, and whose mutations are associated with X-linked ID. Oligophrenin-1 is expressed in neurons, where its functions have been widely reported at the synapse, as well as in glial cells. However, its roles in astrocytes are still largely unexplored. Using in vitro and in vivo models of oligophrenin1 disruption in astrocytes, we found that oligophrenin1 regulates at the molecular level the RhoA/ROCK/MLC2 pathway in astroglial cells. We also showed at the cellular level that oligophrenin1 modulates astrocyte morphology and migration both in vitro and in vivo, and is involved in glial scar formation. Altogether, these data suggest that oligophrenin1 deficiency alters not only neuronal but also astrocytic functions, which might contribute to the development of ID.
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http://dx.doi.org/10.1002/glia.23801DOI Listing
September 2020

Publisher Correction: Progenitors from the central nervous system drive neurogenesis in cancer.

Nature 2020 01;577(7792):E10

UMR967 (Laboratoire Cancer et Microenvironnement ATIP/AVENIR-INSERM-CEA), Institut de Radiobiologie Cellulaire et Moléculaire, Institut de Biologie François Jacob, Direction de la Recherche Fondamentale, Paris, France.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-019-1837-4DOI Listing
January 2020

A proline-rich motif on VGLUT1 reduces synaptic vesicle super-pool and spontaneous release frequency.

Elife 2019 10 30;8. Epub 2019 Oct 30.

Interdisciplinary Institute for Neuroscience, Université de Bordeaux, Bordeaux, France.

Glutamate secretion at excitatory synapses is tightly regulated to allow for the precise tuning of synaptic strength. Vesicular Glutamate Transporters (VGLUT) accumulate glutamate into synaptic vesicles (SV) and thereby regulate quantal size. Further, the number of release sites and the release probability of SVs maybe regulated by the organization of active-zone proteins and SV clusters. In the present work, we uncover a mechanism mediating an increased SV clustering through the interaction of VGLUT1 second proline-rich domain, endophilinA1 and intersectin1. This strengthening of SV clusters results in a combined reduction of axonal SV super-pool size and miniature excitatory events frequency. Our findings support a model in which clustered vesicles are held together through multiple weak interactions between Src homology three and proline-rich domains of synaptic proteins. In mammals, VGLUT1 gained a proline-rich sequence that recruits endophilinA1 and turns the transporter into a regulator of SV organization and spontaneous release.
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http://dx.doi.org/10.7554/eLife.50401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861006PMC
October 2019

The C-terminal domain of LRRK2 with the G2019S mutation is sufficient to produce neurodegeneration of dopaminergic neurons in vivo.

Neurobiol Dis 2020 02 9;134:104614. Epub 2019 Oct 9.

CEA, DRF, Institut de Biologie Françoise Jacob, Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses, France; CNRS, CEA, Paris-Sud Univ., Univ. Paris-Saclay, Neurodegenerative Diseases Laboratory (UMR9199), F-92265 Fontenay-aux-Roses, France. Electronic address:

The G2019S substitution in the kinase domain of LRRK2 (LRRK2) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2 are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2 is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2 in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2 led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2 or LV-ΔLRRK2, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2 could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2 and ΔLRRK2 at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2 produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2 did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.
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http://dx.doi.org/10.1016/j.nbd.2019.104614DOI Listing
February 2020

Structural and functional connections between the median and the ventrolateral preoptic nucleus.

Brain Struct Funct 2019 Dec 6;224(9):3045-3057. Epub 2019 Sep 6.

Neuroglial Interactions in Cerebral Physiopathology, Center for Interdisciplinary Research in Biology, Centre National de la Recherche Scientifique UMR 7241, Institut National de la Santé et de la Recherche Médicale U1050, Collège de France, Labex Memolife, PSL-University, 11 place Marcelin Berthelot, 75005, Paris, France.

The median preoptic nucleus (MnPO) and the ventrolateral preoptic nucleus (VLPO) are two brain structures that contain neurons essential for promoting non-rapid eye movement (NREM) sleep. However, their connections are still largely unknown. Here, we describe for the first time a slice preparation with an oblique coronal slicing angle at 70° from the horizontal in which their connectivity is preserved. Using the in vivo iDISCO method following viral infection of the MnPO or ex vivo biocytin crystal deposition in the MnPO of mouse brain slices, we revealed a strong axonal pathway from the MnPO to the VLPO. Then, to further explore the functionality of these projections, acute 70° slices were placed on multielectrode arrays (MEAs) and electrical stimulations were performed near the MnPO. Recordings of the signals propagation throughout the slices revealed a preferential pathway from the MnPO to the VLPO. Finally, we performed an input-output curve of field responses evoked by stimulation of the MnPO and recorded in the VLPO. We found that field responses were inhibited by GABA receptor antagonist, suggesting that afferent inputs from the MnPO activate VLPO neuronal networks by disinhibition.
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http://dx.doi.org/10.1007/s00429-019-01935-4DOI Listing
December 2019

Progenitors from the central nervous system drive neurogenesis in cancer.

Nature 2019 05 15;569(7758):672-678. Epub 2019 May 15.

UMR967 (Laboratoire Cancer et Microenvironnement ATIP/AVENIR-INSERM-CEA), Institut de Radiobiologie Cellulaire et Moléculaire, Institut de Biologie François Jacob, Direction de la Recherche Fondamentale, Paris, France.

Autonomic nerve fibres in the tumour microenvironment regulate cancer initiation and dissemination, but how nerves emerge in tumours is currently unknown. Here we show that neural progenitors from the central nervous system that express doublecortin (DCX) infiltrate prostate tumours and metastases, in which they initiate neurogenesis. In mouse models of prostate cancer, oscillations of DCX neural progenitors in the subventricular zone-a neurogenic area of the central nervous system-are associated with disruption of the blood-brain barrier, and with the egress of DCX cells into the circulation. These cells then infiltrate and reside in the tumour, and can generate new adrenergic neurons. Selective genetic depletion of DCX cells inhibits the early phases of tumour development in our mouse models of prostate cancer, whereas transplantation of DCX neural progenitors promotes tumour growth and metastasis. In humans, the density of DCX neural progenitors is strongly associated with the aggressiveness and recurrence of prostate adenocarcinoma. These results reveal a unique crosstalk between the central nervous system and prostate tumours, and indicate neural targets for the treatment of cancer.
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http://dx.doi.org/10.1038/s41586-019-1219-yDOI Listing
May 2019

Publisher Correction: Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy.

Nat Commun 2019 May 9;10(1):2160. Epub 2019 May 9.

Laboratory for Optics and Biosciences, Ecole polytechnique, CNRS, INSERM, IP Paris, Palaiseau, 91128, France.

Affiliation 4 incorrectly read 'University of the Basque Country (Ikerbasque), University of the Basque Country and Donostia International Physics Center, San Sebastian 20018, Spain.'Also, the affiliations of Ignacio Arganda-Carreras with 'IKERBASQUE, Basque Foundation for Science, Bilbao, 48013, Spain' and 'Donostia International Physics Center (DIPC), San Sebastian, 20018, Spain' were inadvertently omitted.Additionally, the third sentence of the first paragraph of the Results section entitled 'Multicontrast organ-scale imaging with ChroMS microscopy' incorrectly read 'For example, one can choose lambda1 = 850 and lambda2 = 110 nm for optimal two-photon excitation of blue and red chromophores.'. The correct version reads 'lambda2 = 1100 nm' instead of 'lambda2 = 110 nm'. These errors have now been corrected in the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-019-10225-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509334PMC
May 2019

Multicolor multiscale brain imaging with chromatic multiphoton serial microscopy.

Nat Commun 2019 04 10;10(1):1662. Epub 2019 Apr 10.

Laboratory for Optics and Biosciences, Ecole polytechnique, CNRS, INSERM, IP Paris, Palaiseau, 91128, France.

Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one-shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.
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http://dx.doi.org/10.1038/s41467-019-09552-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458155PMC
April 2019

Author Correction: TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

Nat Commun 2018 08 1;9(1):3109. Epub 2018 Aug 1.

Institute of Biology Paris Seine, Gene Regulation and Adaptive Behaviors Team, Department of Neuroscience Paris Seine, Sorbonne Université, CNRS UMR 8246 & INSERM U1130, 9 Quai Saint Bernard, F-75005, Paris, France.

The originally published version of this Article contained an error in the subheading "Microglial GR does not affect DN loss triggered by TLR4 and TLR7," which was incorrectly given as "Microglial GR does affect DN loss triggered by TLR2 and TLR4". This has now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-05680-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070486PMC
August 2018

TLR9 activation via microglial glucocorticoid receptors contributes to degeneration of midbrain dopamine neurons.

Nat Commun 2018 06 22;9(1):2450. Epub 2018 Jun 22.

Institute of Biology Paris Seine, Gene Regulation and Adaptive Behaviors Team, Department of Neuroscience Paris Seine, Sorbonne Université, CNRS UMR 8246 & INSERM U1130, 9 Quai Saint Bernard, F-75005, Paris, France.

Inflammation is a characteristic feature of Parkinson's disease (PD). We examined the role of TLR9 and its regulation by glucocorticoid receptors (GRs) in degeneration of substantia nigra dopamine neurons (DNs). TLR9 agonist, CpG-ODN, induced DN degeneration in mice lacking GR in microglia but not in controls. TLR9 deletion reduced DN loss in neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. GR regulates TLR9 activation during MPTP neurotoxicity as TLR9 antagonist suppressed increased DN loss in microglia/macrophage GR mutant mice. GR absence in microglia enhanced TLR9 translocation to endolysosomes and facilitated its cleavage leading to pro-inflammatory gene expression. GR-dependent TLR9 activation also triggered DN loss following intranigral injection of mitochondrial DNA. Finally, microglial GR sensitivity to A53T-alpha-synuclein induced DN degeneration as well as decreased microglial GR expression observed in SN of PD brain samples, all suggest that reduced microglial GR activity in SN can stimulate TLR9 activation and DN loss in PD pathology.
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http://dx.doi.org/10.1038/s41467-018-04569-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015079PMC
June 2018

The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin.

Brain 2018 05;141(5):1434-1454

CEA, DRF, Institut François Jacob, Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses, France.

The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington's disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington's disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington's disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington's disease could render the striatum highly vulnerable to neurodegeneration.
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http://dx.doi.org/10.1093/brain/awy057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917821PMC
May 2018

Connexin 30 controls astroglial polarization during postnatal brain development.

Development 2018 02 23;145(4). Epub 2018 Feb 23.

Center for Interdisciplinary Research in Biology, Collège de France, CNRS UMR 7241, INSERM U1050, Labex Memolife, PSL Research University, Paris 75005, France

Astrocytes undergo intense morphological maturation during development, changing from individual sparsely branched cells to polarized and tremendously ramified cells. Connexin 30, an astroglial gap-junction channel-forming protein expressed postnatally, regulates the extension and ramification of astroglial processes. However, the involvement of connexin 30 in astroglial polarization, which is known to control cell morphology, remains unexplored. We found that connexin 30, independently of gap-junction-mediated intercellular biochemical coupling, alters the orientation of astrocyte protrusion, centrosome and Golgi apparatus during polarized migration in an wound-healing assay. Connexin 30 sets the orientation of astroglial motile protrusions via modulation of the laminin/β1 integrin/Cdc42 polarity pathway. Connexin 30 indeed reduces laminin levels, inhibits the redistribution of the β1-integrin extracellular matrix receptors, and inhibits the recruitment and activation of the small Rho GTPase Cdc42 at the leading edge of migrating astrocytes. , connexin 30, the expression of which is developmentally regulated, also contributes to the establishment of hippocampal astrocyte polarity during postnatal maturation. This study thus reveals that connexin 30 controls astroglial polarity during development.
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http://dx.doi.org/10.1242/dev.155275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869003PMC
February 2018

Potentiating tangle formation reduces acute toxicity of soluble tau species in the rat.

Brain 2018 02;141(2):535-549

CEA, DRF, Institut François Jacob, Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses, France.

Tauopathies are neurodegenerative diseases characterized by the aggregation of tau protein. These pathologies exhibit a wide variety of clinical and anatomo-pathological presentations, which may result from different pathological mechanisms. Although tau inclusions are a common feature in all these diseases, recent evidence instead implicates small oligomeric aggregates as drivers of tau-induced toxicity. Hence in vivo model systems displaying either soluble or fibrillary forms of wild-type or mutant tau are needed to better identify their respective pathological pathways. Here we used adeno-associated viruses to mediate gene transfer of human tau to the rat brain to develop models of pure tauopathies. Two different constructs were used, each giving rise to a specific phenotype developing in less than 3 months. First, hTAUWT overexpression led to a strong hyperphosphorylation of the protein, which was associated with neurotoxicity in the absence of any significant aggregation. In sharp contrast, its co-expression with the pro-aggregation peptide TauRD-ΔK280 in the hTAUProAggr group strongly promoted its aggregation into Gallyas-positive neurofibrillary tangles, while preserving neuronal survival. Our results support the hypothesis that soluble tau species are key players of tau-induced neurodegeneration.
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http://dx.doi.org/10.1093/brain/awx342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837551PMC
February 2018

βAPP Processing Drives Gradual Tau Pathology in an Age-Dependent Amyloid Rat Model of Alzheimer's Disease.

Cereb Cortex 2018 11;28(11):3976-3993

INSERM UMR1169, Université Paris-Sud, Université Paris-Saclay, Orsay, France.

The treatment of Alzheimer's disease (AD) remains challenging and requires a better in depth understanding of AD progression. Particularly, the link between amyloid protein precursor (APP) processing and Tau pathology development remains poorly understood. Growing evidences suggest that APP processing and amyloid-β (Aβ) release are upstream of Tau pathology but the lack of animal models mimicking the slow progression of human AD raised questions around this mechanism. Here, we described that an AD-like βAPP processing in adults wild-type rats, yielding to human APP, βCTF and Aβ levels similar to those observed in AD patients, is sufficient to trigger gradual Tauopathy. The Tau hyperphosphorylation begins several months before the formation of both amyloid plaques and tangle-like aggregates in aged rats and without associated inflammation. Based on a longitudinal characterization over 30 months, we showed that extrasynaptic and emotional impairments appear before long-term potentiation deficits and memory decline and so before Aβ and Tau aggregations. These compelling data allowed us to (1) experimentally confirm the causal relationship between βAPP processing and Tau pathology in vivo and without Tau transgene overexpression, (2) support the amyloidogenic cascade and (3) propose a 4-step hypothesis of prodromal AD progression.
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http://dx.doi.org/10.1093/cercor/bhx260DOI Listing
November 2018

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina.

Mol Ther 2017 11 20;25(11):2546-2560. Epub 2017 Jul 20.

INSERM U968, Institut de la Vision, 75012 Paris, France; UMRS968, Institut de la Vision, Sorbonne Universités, Pierre et Marie Curie University (UPMC) University Paris 06, 75012 Paris, France; Centre National de la Recherche Scientifique (CNRS) UMR7210, Institut de la Vision, 75012 Paris, France. Electronic address:

The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second- and third-order neurons are spared in these diseases, making it possible to restore retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca-permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus [CMV]) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675708PMC
November 2017

Evaluation of tolerance to lentiviral LV-RPE65 gene therapy vector after subretinal delivery in non-human primates.

Transl Res 2017 10 8;188:40-57.e4. Epub 2017 Jul 8.

Unit of Retinal Degeneration and Regeneration, Department of Ophthalmology, University of Lausanne, Jules-Gonin Eye Hospital, Fondation Asile des Aveugles, Lausanne, Switzerland. Electronic address:

Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, strategies that have reached the clinical stages rely on adeno-associated viral vectors and two of them documented limited long-term effect. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restored protein expression and cone function in RPE65-deficient mice. In this study, we evaluated the ocular and systemic tolerances of this lentiviral-based therapy (LV-RPE65) on healthy nonhuman primates (NHPs), without adjuvant systemic anti-inflammatory prophylaxis. For the first time, we describe the early kinetics of retinal detachment at 2, 4, and 7 days after subretinal injection using multimodal imaging in 5 NHPs. We revealed prolonged reattachment times in LV-RPE65-injected eyes compared to vehicle-injected eyes. Low- (n = 2) and high-dose (n = 2) LV-RPE65-injected eyes presented a reduction of the outer nuclear and photoreceptor outer segment layer thickness in the macula, that was more pronounced than in vehicle-injected eyes (n = 4). All LV-RPE65-injected eyes showed an initial perivascular reaction that resolved spontaneously within 14 days. Despite foveal structural changes, full-field electroretinography indicated that the overall retinal function was preserved over time and immunohistochemistry identified no difference in glial, microglial, or leucocyte ocular activation between low-dose, high-dose, and vehicle-injected eyes. Moreover, LV-RPE65-injected animals did not show signs of vector shedding or extraocular targeting, confirming the safe ocular restriction of the vector. Our results evidence a limited ocular tolerance to LV-RPE65 after subretinal injection without adjuvant anti-inflammatory prophylaxis, with complications linked to this route of administration necessitating to block this transient inflammatory event.
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http://dx.doi.org/10.1016/j.trsl.2017.06.012DOI Listing
October 2017

Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8.

Biotechnol Bioeng 2016 12 30;113(12):2712-2724. Epub 2016 Jun 30.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, 17 rue Moreau, Paris 75012, France.

Recently, we described a modified AAV2 vector-AAV2-7m8-having a capsid-displayed peptide insertion of 10 amino acids with enhanced retinal transduction properties. The insertion of the peptide referred to as 7m8 is responsible for high-level gene delivery into deep layers of the retina when virus is delivered into the eye's vitreous. Here, we further characterize AAV2-7m8 mediated gene delivery to neural tissue and investigate the mechanisms by which the inserted peptide provides better transduction away from the injection site. First, in order to understand if the peptide exerts its effect on its own or in conjunction with the neighboring amino acids, we inserted the 7m8 peptide at equivalent positions on three other AAV capsids, AAV5, AAV8, and AAV9, and evaluated its effect on their infectivity. Intravitreal delivery of these peptide insertion vectors revealed that only AAV9 benefited from 7m8 insertion in the context of the retina. We then investigated AAV2-7m8 and AAV9-7m8 properties in the brain, to better evaluate the spread and efficacy of viral transduction in view of the peptide insertion. While 7m8 insertion led to higher intensity gene expression, the spread of gene expression remained unchanged compared to the parental serotypes. Our results indicate that the 7m8 peptide insertion acts by increasing efficacy of cellular entry, with little effect on the spread of viral particles in neural tissue. The effects of peptide insertion are capsid and tissue dependent, highlighting the importance of the microenvironment in gene delivery using AAV. Biotechnol. Bioeng. 2016;113: 2712-2724. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/bit.26031DOI Listing
December 2016

Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain.

Sci Rep 2016 06 20;6:28272. Epub 2016 Jun 20.

Schaller Research Group at the University of Heidelberg and the German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany.

Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer's disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.
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http://dx.doi.org/10.1038/srep28272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913310PMC
June 2016

mRNA trans-splicing in gene therapy for genetic diseases.

Wiley Interdiscip Rev RNA 2016 07 28;7(4):487-98. Epub 2016 Mar 28.

Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Département des Sciences du Vivant (DSV), Institut d'Imagerie Biomédicale (I2BM), Molecular Imaging Research Center (MIRCen), Fontenay-aux-Roses, France.

Spliceosome-mediated RNA trans-splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post-transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre-mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans-splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans-splicing, review the different strategies that are under evaluation to lead to efficient trans-splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487-498. doi: 10.1002/wrna.1347 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wrna.1347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5071737PMC
July 2016

Alzheimer's disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression.

Mol Neurodegener 2016 Jan 12;11. Epub 2016 Jan 12.

INSERM UMR1169, Université Paris-Sud, Université Paris-Saclay, Orsay, 94100, France.

Background: Alzheimer's disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing.

Results: The human APP, βCTF and Aβ42/40 ratio were similar to those in hippocampal tissues from AD patients. Three months after injection the murine Tau protein was hyperphosphorylated and rapid synaptic failure occurred characterized by decreased levels of both PSD-95 and metabolites related to neuromodulation, on proton magnetic resonance spectroscopy ((1)H-MRS). Astrocytic GLT-1 transporter levels were lower and the tonic glutamatergic current was stronger on electrophysiological recordings of CA1 hippocampal region, revealing the overstimulation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) which precedes the loss of long-term potentiation (LTP). These modifications were associated with early behavioral impairments in the Open-field, Y-maze and Morris Mater Maze tasks.

Conclusions: Altogether, this demonstrates that an AD-like APP processing, yielding to levels of APP, βCTF and Aβ42/Aβ40 ratio similar to those observed in AD patients, are sufficient to rapidly trigger early steps of the amyloidogenic and Tau pathways in vivo. With this strategy, we identified a sequence of early events likely to account for disease onset and described a model that may facilitate efforts to decipher the factors triggering AD and to evaluate early neuroprotective strategies.
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http://dx.doi.org/10.1186/s13024-016-0070-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709894PMC
January 2016

Viral gene transfer of APPsα rescues synaptic failure in an Alzheimer's disease mouse model.

Acta Neuropathol 2016 Feb 4;131(2):247-266. Epub 2015 Nov 4.

Department of Bioinformatics and Functional Genomics, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120, Heidelberg, Germany.

Alzheimer's disease (AD) is characterized by synaptic failure, dendritic and axonal atrophy, neuronal death and progressive loss of cognitive functions. It is commonly assumed that these deficits arise due to β-amyloid accumulation and plaque deposition. However, increasing evidence indicates that loss of physiological APP functions mediated predominantly by neurotrophic APPsα produced in the non-amyloidogenic α-secretase pathway may contribute to AD pathogenesis. Upregulation of APPsα production via induction of α-secretase might, however, be problematic as this may also affect substrates implicated in tumorigenesis. Here, we used a gene therapy approach to directly overexpress APPsα in the brain using AAV-mediated gene transfer and explored its potential to rescue structural, electrophysiological and behavioral deficits in APP/PS1∆E9 AD model mice. Sustained APPsα overexpression in aged mice with already preexisting pathology and amyloidosis restored synaptic plasticity and partially rescued spine density deficits. Importantly, AAV-APPsα treatment also resulted in a functional rescue of spatial reference memory in the Morris water maze. Moreover, we demonstrate a significant reduction of soluble Aβ species and plaque load. In addition, APPsα induced the recruitment of microglia with a ramified morphology into the vicinity of plaques and upregulated IDE and TREM2 expression suggesting enhanced plaque clearance. Collectively, these data indicate that APPsα can mitigate synaptic and cognitive deficits, despite established pathology. Increasing APPsα may therefore be of therapeutic relevance for AD.
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http://dx.doi.org/10.1007/s00401-015-1498-9DOI Listing
February 2016

Deciphering the molecular basis of invasiveness in Sdhb-deficient cells.

Oncotarget 2015 Oct;6(32):32955-65

INSERM, UMR970, Paris Cardiovascular Research Centre, F-75015 Paris, France.

Metastatic pheochromocytomas and paragangliomas (PPGL) are malignant neuroendocrine tumors frequently associated with germline mutations in the SDHB gene. SDHB-mutated PPGL display a hypermethylator phenotype associated with hallmarks of epithelial-to-mesenchymal transition (EMT). In the present study, we report the characterization of a unique model of Sdhb knockout in mouse chromaffin cells. Sdhb deficient cells exhibit a metastatic phenotype as highlighted by increased individual cell migration (characterized by faster motility and increased persistence) as well as high invasive and adhesion abilities. This phenotype is associated with the modulation of Twist1, Twist2, Tcf3, Snai1, N-cadherin or Krt19 expression, reflecting an EMT-like reprogramming of cells. Krt19 is epigenetically silenced in Sdhb-deficient cells and re-expressed after treatment by the demethylating agent decitabine. Krt19 rescue by lentiviral transduction in Sdhb-deficient cells and Krt19 inhibition by RNA interference in wild-type cells were performed. Both studies revealed the involvement of KRT19 in the invasive phenotype by modulating collective and individual migration and cell/extra-cellular matrix adhesion properties. These findings underline the role of hypermethylation and EMT in the in vitro acquisition of metastatic properties, following SDHB loss of function.
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http://dx.doi.org/10.18632/oncotarget.5106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741742PMC
October 2015
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