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See detailElp3 Lysine Acetyl-Transferase Controls Neuronal Survival in the Developing Inner Ear and is Crucial to Balance and Hearing
Delacroix, Laurence ULg; Mateo Sanchez, Susana ULg; Freeman, Stephen ULg et al

Conference (2016, February 20)

Elp3 lysine acetyl-transferase, the catalytic subunit of the Elongator complex, has been assigned multiple roles in gene transcription, DNA methylation and protein translation efficiency. Given the ... [more ▼]

Elp3 lysine acetyl-transferase, the catalytic subunit of the Elongator complex, has been assigned multiple roles in gene transcription, DNA methylation and protein translation efficiency. Given the importance of acetylation homeostasis in controlling developmental processes together with recent reports implicating Elp3 in cortical neurogenesis, we investigated its role during inner ear formation. In the inner ear, we detected Elp3 transcript in the sensory epithelia of the entire otic vesicle at embryonic day E11.5. At later stages, Elp3 mRNA is strongly expressed in the vestibular and spiral ganglion neurons. To investigate the role of Elp3 in vivo, we used a conditional knock-out mice (Foxg1Cre) in which the expression of the acetyl-transferase is lost in early otocyst. These mice show obvious vestibular defects as indicated by a stereotyped circling ambulation, head bobbing, retropulsion and the absence of a reaching response in the tail-hanging test. Furthermore, we identified a severe hearing loss in Elp3cKO mice through Auditory Brainstem Responses. We show that Elp3 enzyme is crucial for neuronal survival in the spiral ganglion and in the vestibule and that it ensures a correct innervation pattern in the developing inner ear. In the absence of Elp3, a drastic increase in the number of apoptotic neurons was detected by active Caspase-3 and pH2AX immunostainings, particularly during the early stages of development (between E12.5 and E14.5). Postnatally, the neurons remaining in Elp3cKO cochleae seem to establish synaptic contacts with the sensory cells but show obvious signs of cell damage as evidenced by Transmission Electron Microscopy. Taken together, these data support a role for Elp3 in hearing and balance and point out an important role for acetylation homeostasis during inner ear formation. We are currently investigating the molecular mechanisms underlying Elp3 effect on neuronal survival and pathfinding. [less ▲]

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See detailUnravelling the roles of lysine acetylation by Elp3 during inner ear development
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Freeman, Stephen ULg et al

Poster (2016, January 25)

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed ... [more ▼]

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea, in the spiral ganglion and in the vestibule. To unravel functions of Elp3, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3cKO). We submitted these mice to a battery of vestibular testing and found significant abnormalities. Besides, the auditory brain stem response of Elp3cKO indicated that these mice are severely deaf. We were also able to demonstrate an increased level of apoptosis in the Elp3cKO spiral ganglion leading to a reduced number of neurons and fibers innervating the sensory cells as well as a reduced number of their synaptic ribbons. Moreover, the remaining spiral ganglion neurons extend processes showing clearly defects regarding sensory cell innervation. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

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See detailUNRAVELLING THE ROLES OF LYSINE ACETYLATION BY ELP3 DURING INNER EAR DEVELOPMENT
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Freeman, Stephen ULg et al

Poster (2015, November 23)

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed ... [more ▼]

We planned to investigate the role of Elp3 acetyl-transferase, a member of the Elongator complex, in inner ear formation. We first analysed the spatio-temporal pattern of Elp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea, in the spiral ganglion and in the vestibule. To unravel functions of Elp3, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3cKO). We submitted these mice to a battery of vestibular testing and found significant abnormalities. Besides, the auditory brain stem response of Elp3cKO indicated that these mice are severely deaf. We were also able to demonstrate an increased level of apoptosis in the Elp3cKO spiral ganglion leading to a reduced number of neurons and fibers innervating the sensory cells as well as a reduced number of their synaptic ribbons. Moreover, the remaining spiral ganglion neurons extend processes showing clearly defects regarding sensory cell innervation. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

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See detailUNRAVELLING THE ROLES OF LYSINE ACETYLATION BY ELP3 DURING INNER EAR DEVELOPMENT
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Freeman, Stephen ULg et al

Poster (2015, June 06)

Given the importance of acetylation homeostasis in controlling developmental processes [1-3], we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase ... [more ▼]

Given the importance of acetylation homeostasis in controlling developmental processes [1-3], we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis [4]. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of neurons and fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P15. Moreover, the remaining spiral ganglion neurons extend processes showing clearly defects regarding hair cells innervation (misorientation of fibers). In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

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See detailUnravelling Cemip expression and functions in the auditory portion of the inner ear.
Czajkowski, Amandine ULg; Chariot, Alain ULg; Delacroix, Laurence ULg et al

Poster (2015, May 29)

The inner ear is a complex organ composed of the vestibular system – which is the balancing system – and the cochlea – which is the earing system. The cochlea is a coiled shape organ composed of three ... [more ▼]

The inner ear is a complex organ composed of the vestibular system – which is the balancing system – and the cochlea – which is the earing system. The cochlea is a coiled shape organ composed of three main structures: the spiral ligament sitting on top of the stria vascularis, the organ of Corti with sensory hair cells and supporting cells and the spiral ganglion composed of neurons and glial cells. After an auditory stimulus, the sound wave progresses in the scala media filled with endolymph and induces a stimulation of sensory hair cells. These cells then transmit the information to the spiral ganglion neurons connected to them. Of course, the correct ionic homeostasis of endolymph is required for a good sound wave transmission. This homeostatic function is assured by the stria vascularis and the spiral ligament. The alteration of one of the structures mentioned before induces deafness. Currently, numerous genes have been associated to this kind of hearing loss. In the present work, we focus our attention Cemip – also known as KIAA1199 – that has been associated to human hereditary neurosensory deafness. Indeed, three missense mutations consisting in non-synonymous amino acid changes (R187L, R187H and H783Y) have been associated to this form of deafness. Therefore we would like to understand the role of Cemip in the cochlea. For that we have analysed Cemip mRNA pattern of expression by in situ hybridization at different developmental stages on cochlear sections. It seems Cemip mRNA is not present in the auditory portion of the inner ear at early embryonic stage 14 (E14) while it is largely present at E17 in the spiral ganglion, in supporting cells of the organ of Corti and in the spiral ligament. This expression is maintained post-nattily until P7. At P21 the expression is restricted to the spiral lamina - an osseous structure surrounding the spiral ganglion. Our on going work is aimed at revealing the biological role of Cemip in the cochlea in conditional knock-out mice. [less ▲]

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See detailMicroRNA-124 Regulates Cell Specification in the Cochlea through Modulation of Sfrp4/5.
Huyghe, Aurelia; Van Den Ackerveken, Priscilla ULg; SACHELI, Rosalie ULg et al

in Cell Reports (2015), 13

The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the ... [more ▼]

The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the specification of these progenitors remain elusive. In the present study, by combining microarray analyses with conditional deletion of Dicer in the developing inner ear, we identified that miR-124 controls cell fate in the developing organ of Corti. By targeting secreted frizzled-related protein 4 (Sfrp4) and Sfrp5, two inhibitors of the Wnt pathway, we showed that miR-124 controls the β-catenin-dependent and also the PCP-related non-canonical Wnt pathways that contribute to HC differentiation and polarization in the organ of Corti. Thus, our work emphasizes the importance of miR-124 as an epigenetic safeguard that fine-tunes the expression of genes critical for cell patterning during cochlear differentiation. [less ▲]

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See detailUnravelling the roles of lysine acetylation by Elp3 during inner ear development
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Laguesse, Sophie et al

Conference (2014, January 27)

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a ... [more ▼]

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons P15. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

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See detailChapter 7 – Neuronal Circuitries During Inner Ear Development
defourny, jean; Delacroix, Laurence ULg; Malgrange, Brigitte ULg

in Romand, raymond; Varela-Nieto, Isabel (Eds.) Development of auditory and vestibular systems (2014)

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See detailUnravelling the roles of lysine acetyl-transferase activity of Elongator complex during inner ear development
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Laguesse, Sophie et al

Conference (2014)

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a ... [more ▼]

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P15. To find new potential targets for Elp3, transcriptomes from wild-type, heterozygous and Elp3 cKO mice were analysed by RNA-Seq at E14.5 and E18.5. Surprisingly, we observed that hair cell markers were upregulated in the Elp3 cKO at E14.5, suggesting a premature differentiation in these mice that was confirmed by in situ hybridisation. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

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See detailUnravelling the roles of lysine acetylation by Elp3 during inner ear development
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Laguesse, Sophie et al

Poster (2013, October 18)

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a ... [more ▼]

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of Elp3 in the inner ear, we first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons P15. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

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See detailUnravelling the roles of lysine acetylation by Elp3 during inner ear development
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Laguesse, Sophie ULg et al

Poster (2013, May 31)

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a ... [more ▼]

Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. We first analysed the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea, the spiral ganglion, the stria vascularis and the vestibule. To unravel functions of Elp3 in the inner ear, we used conditional knock-out mice in which Elp3 gene is deleted from early otocyst (Elp3 cKO). We submitted these mice to a battery of vestibular testing and found significant abnormalities. Besides, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we detected some defaults in the planar orientation of the auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons. In conclusion, our results clearly show a role for Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by Elp3. [less ▲]

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See detailUnravelling the roles of lysine acetylation by Elp3 during inner ear development
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Laguesse, Sophie ULg et al

Poster (2013, January 28)

The inner ear is composed of the vestibular system that controls balance, and the cochlea, which is dedicated to hearing. In both parts of the inner ear, sensory epithelia comprise supporting cells ... [more ▼]

The inner ear is composed of the vestibular system that controls balance, and the cochlea, which is dedicated to hearing. In both parts of the inner ear, sensory epithelia comprise supporting cells surrounding the sensory hair cells. These cells bear at their apical surface a staircase-structured bundle, consisting of multiple rows of actin-based stereocilia and a single tubulin-based kinocilium. This hair bundle allows the transduction from mechanical stimuli, initiated by sound or gravitational changes, to electrical signals that will then be transmitted by neurons from the spiral ganglion (innervating hair cells of the cochlea) or the vestibular ganglion. The inner ear organogenesis requires a tightly regulated transcriptional program that can be affected by post-transcriptional and post-translational modifications among which lysine acetylation. Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of Elp3 in the inner ear, we first determine the spatio-temporal pattern of ELp3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of Elp3 in the inner ear, we have generated conditional knock-out mice (Elp3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, compared to wild-type mice, the auditory brain stem response of Elp3 cKO indicated that these mice are severely deaf. At the cellular level, we did not found any structural abnormalities nor cell patterning impairments that could explain deafness or balance dysfunction in Elp3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from Elp3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P0 and P15. In conclusion, our results clearly showed a role of Elp3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins acetylated by Elp3. [less ▲]

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See detailGene transfer in inner ear cells: a challenging race
Sacheli, Rosalie ULg; Delacroix, Laurence ULg; Van Den Ackerveken, Priscilla ULg et al

in Gene Therapy (2013), 20

Recent advances in human genomics led to the identification of numerous defective genes causing deafness, which represent novel putative therapeutic targets. Future gene-based treatment of deafness ... [more ▼]

Recent advances in human genomics led to the identification of numerous defective genes causing deafness, which represent novel putative therapeutic targets. Future gene-based treatment of deafness resulting from genetic or acquired sensorineural hearing loss may include strategies ranging from gene therapy to antisense delivery. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene carrier systems. Transfer of exogenous genetic material into the mammalian inner ear using viral or non-viral vectors has been characterized over the last decade. The nature of inner ear cells targeted, as well as the transgene expression level and duration, are highly dependent on the vector type, the route of administration and the strength of the promoter driving expression. This review summarizes and discusses recent advances in inner ear gene-transfer technologies aimed at examining gene function or identifying new treatment for inner ear disorders. [less ▲]

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See detailEvaluating Effects of Tyrosine Phosphatase Inhibitors on T Cell Receptor Signaling
Rahmouni, Souad ULg; Delacroix, Laurence ULg; Liu, Wallace et al

in Phosphatase Modulators, Methods in Molecular Biology (2013)

The importance of tyrosine phosphorylation in normal cell physiology is well established, highlighted by the many human diseases that stem from abnormalities in protein tyrosine kinase (PTK) and protein ... [more ▼]

The importance of tyrosine phosphorylation in normal cell physiology is well established, highlighted by the many human diseases that stem from abnormalities in protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) function. Contrary to earlier assumptions, it is now clear that both PTKs and PTPs are highly specific, non-redundant, and tightly regulated enzymes. Hematopoietic cells express particularly high numbers of PTKs and PTPs, and aberrant function of these proteins have been linked to many hematopoietic disorders. While PTK inhibitors are among FDA approved drugs for the treatment of leukemia and other cancers, efforts to develop therapeutics that target specific PTPs are still in its infancy. Here, we describe methods on how to evaluate effects of PTP inhibitors on T cell receptor signaling. Moreover, we provide a comprehensive strategy for compound prioritization, applicable to any drug discovery project involving T cells. We present a testing funnel that starts with relatively high-throughput luciferase reporter assays, followed by immunoblot, calcium flux, flow cytometry, and proliferation assays, continues with cytokine bead arrays, and finishes with specificity assays that involve RNA interference. We provide protocols for experiments in the Jurkat T cell line, but more importantly give detailed instructions, paired with numerous tips, on how to prepare and work with primary human T cells. [less ▲]

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See detailRetinoic acid receptors recognise the mouse genome through binding elements with diverse spacing and topology
Moutier, Emmanuel; Ye, Tao; Choukrallah, Mohamed-Amin et al

in Journal of Biological Chemistry (2012)

Retinoic Acid Receptors (RARs) heterodimerise with Retinoid X Receptors (RXRs) and bind to RA-response elements (RAREs) in the regulatory regions of their target genes. While previous studies on limited ... [more ▼]

Retinoic Acid Receptors (RARs) heterodimerise with Retinoid X Receptors (RXRs) and bind to RA-response elements (RAREs) in the regulatory regions of their target genes. While previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2 or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8 and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half sites with DR2 and DR0 spacings. This specific half site organisation constitutes a previously unrecognised, but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, while DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5 and DR8 to mediate RA-dependent transcriptional activation indicates that half site spacing allosterically regulates RAR function. [less ▲]

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See detailUnravelling the roles of lysine acetylation by Elp3 during inner ear development
Mateo Sanchez, Susana ULg; Delacroix, Laurence ULg; Laguesse, Sophie ULg et al

Poster (2012, May 04)

The inner ear is composed of a vestibular part that controls balance, and the cochlea, which is dedicated to hearing. In both parts of the inner ear, sensory epithelia comprise supporting cells ... [more ▼]

The inner ear is composed of a vestibular part that controls balance, and the cochlea, which is dedicated to hearing. In both parts of the inner ear, sensory epithelia comprise supporting cells surrounding the sensory hair cells. These cells bear at their apical surface a staircase-structured hair bundle, consisting of multiple rows of actin-based stereocilia and a single tubulin-based kinocilium. This hair bundle allows the transduction from mechanical stimuli, initiated by sound or gravitational changes, to electrical signals that will then be transmitted by neurons from the spiral ganglion (innervating hair cells of the cochlea) or the vestibular ganglion. The inner ear organogenesis requires a tightly regulated transcriptional program that can be affected by post-transcriptional and post-translational modifications among which lysine acetylation. Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on Elp3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. First, we have analysed Elp3 expression by in situ hybridization on wild type mice at different developmental stages (from E11.5 until P6) and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the stria vascularis and in the vestibule. To study the functional consequences of protein acetylation by the Elongator complex in the inner ear, we studied conditional knock-out mice (Elp3 cKO) in which Elp3 is depleted from the otic vesicle at E8.5. These mice, at stage P15, showed obvious balance dysfunction that was confirmed by a complete battery of behavioural tests: stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test. Unfortunately, the Elp3 cKO mice die before the onset of hearing, thus precluding any evaluation of hearing disorders. Balance defects in mice depleted for Elp3 is not due to vestibular structural abnormalities, since paint-filling experiments showed a normal inner ear anatomy compared to wild type mice. Moreover, immunostainings in the vestibule and in the organ of Corti indicated that cell patterning was not impaired in the absence of Elp3 since specialised cells are present and correctly organised at embryonic day E18.5 and later on. However, we were able to detect some defaults in hair cell bundle integrity and orientation in the auditory portion of inner ear from Elp3 cKO mice. We were also able to demonstrate an increased level of apoptosis in the Elp3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells at P0 and P15. In conclusion, we have confirmed the expression of Elp3 in the inner ear and pointed out a role for this acetyl-transferase in balance function. Our results clearly show the implication of Elp3 in ciliogenesis, hair cell innervation and neuronal survival and we plan to go deeper in the mechanisms involved through the identification of the proteins acetylated by Elp3. [less ▲]

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See detailDynamic interaction between lymphoid tyrosine phosphatase and C-terminal Src kinase controls T cell activation
Tautz, Lutz; Vang, Torkel; Liu, Wallace et al

in FASEB Journal (2012, April), 26

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T cell antigen receptor (TCR) and are thought to act in a cooperative manner ... [more ▼]

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here, we show that dissociation of the LYP/CSK complex is necessary for recruitment of LYP to lipid rafts, where it down-modulates TCR-mediated signaling. Our findings may also explain the reduced TCR signaling associated with a single nucleotide polymorphism, which confers increased risk for autoimmunity and results in the expression of a LYP allele that can no longer bind CSK. Development of a potent and selective chemical probe of LYP allowed us to confirm that the observed down-modulation of TCR-induced signaling was due to the LYP catalytic activity. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity. [less ▲]

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See detailAn improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rgammanull mice allows HIV replication and development of anti-HIV immune responses.
Singh, Maneesh; Singh, Pratibha; Gaudray, Gilles et al

in PLoS ONE (2012), 7(6), 38491

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rgamma(null) (NSG) and NOD/SCID/IL2Rgamma(null) (NOG) mice need efficient human cell engraftment for long ... [more ▼]

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rgamma(null) (NSG) and NOD/SCID/IL2Rgamma(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials. [less ▲]

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See detailLYP inhibits T-cell activation when dissociated from CSK
Vang; Liu, Wallace H; Delacroix, Laurence ULg et al

in Nature Chemical Biology (2012)

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner ... [more ▼]

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here we studied the spatiotemporal dynamics of the LYP–CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it downmodulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T-cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single-nucleotide polymorphism that confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a mutant LYP that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity. [less ▲]

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