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Urban Lendahl - One of the best experts on this subject based on the ideXlab platform.
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mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase
Journal of Biological Chemistry, 2001Co-Authors: Cecilia Sahlgren, Andrey Mikhailov, Yinghao Chou, Jukka Hellman, Urban Lendahl, Robert D. Goldman, John E. ErikssonAbstract:Abstract The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and 32P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr316 as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr316, which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.
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nestin expression in embryonic and adult human teeth under normal and pathological conditions
American Journal of Pathology, 2000Co-Authors: Imad About, Dominique Laurentmaquin, Urban Lendahl, Thimios A. MitsiadisAbstract:Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivo distribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitro assay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation.
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the intermediate filament protein nestin occurs transiently in differentiating testis of rat and mouse
Differentiation, 1997Co-Authors: K Frojdman, Urban Lendahl, Lauri J Pelliniemi, Ismo Virtanen, John E. ErikssonAbstract:Nestin is an intermediate filament (IF) protein (IFP) which occurs during early developmental stages and during regenerative processes in muscle and neuronal cells. The spatial and temporal localization of nestin in the developing testis of rat and mouse was studied by immunolabeling light and electron microscopy and by immunoblotting. Nestin localization was related to the localization of the other major IFPs specific for this tissue, i.e. cytokeratins, vimentin and desmin. Laminin immunocytochemistry and conventional microscopy were used to identify tissues and cells. With the incipient differentiation of the gonadal anlage, the reaction for nestin was weak in the gonadal ridge, whereas the cells of the mesonephric mesenchyme showed a prominent reaction for this IFP. The nestin-specific reaction in the epithelial mesonephric duct and tubules was weak and disappeared at an early phase of differentiation. With the development of the testis proper, nestin was transiently found in several cell types. Nestin was found as well as vimentin and cytokeratins in the Sertoli cells. In the interstitial cells nestin was found together with vimentin and desmin IFPs, and was most prominent in the differentiating myoid cells. After birth, nestin gradually disappeared from the testicular cells and in the rat at puberty was found only in the endothelial cells of some blood vessels. The abolished nestin synthesis in the testis was confirmed by immunoblotting. These results suggest that nestin is required transiently during the development of the testis and mesonephros. The temporary presence of nestin, and several other IFPs during these phases, coincides with key phases of urogenital sex differentiation. This may imply that the orchestrated synthesis of the IFPs nestin, cytokeratins, vimentin and desmin is likely to be linked with the genes regulating sex differentiation.
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the embryonic intermediate filament protein nestin is present in the cerebrospinal fluid of nicu infants 2223
Pediatric Research, 1996Co-Authors: Mats Blennow, Urban Lendahl, Giedre Grigelioniene, Carina Torok, Hugo LagercrantzAbstract:Nestin is an intermediate filament protein that is predominantly expressed by progenitor cells during early CNS development. Its expression has been suggested to be most prominent during early gestation. However, contradictory results reporting nestin expression in the mature CNS have also been published. We wanted to investigate whether nestin is detectable in the CSF of newborn infants, and if so, whether this expression depends on gestational age and/or various cerebral diseases. Using western blot analysis and densitometry, we examined CSF samples from 20 newborn infants varying from 25 to 42 weeks of gestation. The infants suffered from perinatal asphyxia (n=7), periventricular leukomalacia and periventricular haemorrhage (n=6) and seven infants had no known cerebral disease (n=7). Protein extract from the periventricular brain tissue of a full-term 1-week old infant, who died from congenital heart disease, was also analyzed. Nestin was detected in all the CSF samples and also in the protein extract from brain tissue. A decrease in the relative nestin levels with increasing gestational age was found. There was no correlation between nestin levels and CNS injury. An unexpected finding was that brain-derived nestin had an apparent molecular weight of appx 240 kDa whereas all analysed CSF samples contained two nestin immunoreactive proteins at 200 and 220 kDa. Experimental deglycosylation of the 240 kDa form reduced its MW to 220 kDa, indicating that nestin undergoes a specific deglycosylation upon release into the CSF. The presence of nestin in the CSF and in the brain tissue suggest a population of nestin-positive stem cells in the brain at full-term gestation. The data do not support the use of nestin in the CSF as a marker for perinatal brain damage, but suggest nestin as a potential marker for neuronal progenitor cells.
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rapid widespread and longlasting induction of nestin contributes to the generation of glial scar tissue after cns injury
Journal of Cell Biology, 1995Co-Authors: Jonas Frisén, C Torok, Clas B Johansson, Mårten Risling, Urban LendahlAbstract:Neuronal regeneration does generally not occur in the central nervous system (CNS) after injury, which has been attributed to the generation of glial scar tissue. In this report we show that the composition of the glial scar after traumatic CNS injury in rat and mouse is more complex than previously assumed: expression of the intermediate filament nestin is induced in reactive astrocytes. Nestin induction occurs within 48 hours in the spinal cord both at the site of lesion and in degenerating tracts and lasts for at least 13 months. Nestin expression is induced with similar kinetics in the crushed optic nerve. In addition to the expression in reactive astrocytes, we also observed nestin induction within 48 hours after injury in cells close to the central canal in the spinal cord, while nestin expressing cells at later timepoints were found progressively further out from the central canal. This dynamic pattern of nestin induction after injury was mimicked by lacZ expressing cells in nestin promoter/lacZ transgenic mice, suggesting that defined nestin regulatory regions mediate the injury response. We discuss the possibility that the spatiotemporal pattern of nestin expression reflects a population of nestin positive cells, which proliferates and migrates from a region close to the central canal to the site of lesion in response to injury.
John E. Eriksson - One of the best experts on this subject based on the ideXlab platform.
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a nestin scaffold links cdk5 p35 signaling to oxidant induced cell death
The EMBO Journal, 2006Co-Authors: Cecilia Sahlgren, Yinghao Chou, Hanna-mari Pallari, Tao He, Robert D. Goldman, John E. ErikssonAbstract:The intermediate filament protein, nestin, has been implicated as an organizer of survival-determining signaling molecules. When nestin expression was related to the sensitivity of neural progenitor cells to oxidant-induced apoptosis, nestin displayed a distinct cytoprotective effect. Oxidative stress in neuronal precursor cells led to downregulation of nestin with subsequent activation of cyclin-dependent kinase 5 (Cdk5), a crucial kinase in the nervous system. Nestin downregulation was a prerequisite for the Cdk5-dependent apoptosis, as overexpression of nestin efficiently inhibited induction of apoptosis, whereas depletion of nestin by RNA interference had a sensitizing effect. When the underlying link between nestin and Cdk5 was analyzed, we observed that nestin serves as a scaffold for Cdk5, with binding restricted to a specific region following the alpha-helical domain of nestin, and that the presence and organization of nestin regulated the sequestration and activity of Cdk5, as well as the ubiquitylation and turnover of its regulator, p35. Our data imply that nestin is a survival determinant whose action is based upon a novel mode of Cdk5 regulation, affecting the targeting, activity, and turnover of the Cdk5/p35 signaling complex.
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cdk5 regulates the organization of nestin and its association with p35
Molecular and Cellular Biology, 2003Co-Authors: Cecilia Sahlgren, Andrey Mikhailov, Hanna-mari Pallari, Samuli Vaittinen, Harish C. Pant, Hannu Kalimo, John E. ErikssonAbstract:The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity—either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor—blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. InteRestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.
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mitotic reorganization of the intermediate filament protein nestin involves phosphorylation by cdc2 kinase
Journal of Biological Chemistry, 2001Co-Authors: Cecilia Sahlgren, Andrey Mikhailov, Yinghao Chou, Jukka Hellman, Urban Lendahl, Robert D. Goldman, John E. ErikssonAbstract:Abstract The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and 32P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr316 as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr316, which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.
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the intermediate filament protein nestin occurs transiently in differentiating testis of rat and mouse
Differentiation, 1997Co-Authors: K Frojdman, Urban Lendahl, Lauri J Pelliniemi, Ismo Virtanen, John E. ErikssonAbstract:Nestin is an intermediate filament (IF) protein (IFP) which occurs during early developmental stages and during regenerative processes in muscle and neuronal cells. The spatial and temporal localization of nestin in the developing testis of rat and mouse was studied by immunolabeling light and electron microscopy and by immunoblotting. Nestin localization was related to the localization of the other major IFPs specific for this tissue, i.e. cytokeratins, vimentin and desmin. Laminin immunocytochemistry and conventional microscopy were used to identify tissues and cells. With the incipient differentiation of the gonadal anlage, the reaction for nestin was weak in the gonadal ridge, whereas the cells of the mesonephric mesenchyme showed a prominent reaction for this IFP. The nestin-specific reaction in the epithelial mesonephric duct and tubules was weak and disappeared at an early phase of differentiation. With the development of the testis proper, nestin was transiently found in several cell types. Nestin was found as well as vimentin and cytokeratins in the Sertoli cells. In the interstitial cells nestin was found together with vimentin and desmin IFPs, and was most prominent in the differentiating myoid cells. After birth, nestin gradually disappeared from the testicular cells and in the rat at puberty was found only in the endothelial cells of some blood vessels. The abolished nestin synthesis in the testis was confirmed by immunoblotting. These results suggest that nestin is required transiently during the development of the testis and mesonephros. The temporary presence of nestin, and several other IFPs during these phases, coincides with key phases of urogenital sex differentiation. This may imply that the orchestrated synthesis of the IFPs nestin, cytokeratins, vimentin and desmin is likely to be linked with the genes regulating sex differentiation.
Kuanghung Cheng - One of the best experts on this subject based on the ideXlab platform.
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stem cell marker nestin is critical for tgf β1 mediated tumor progression in pancreatic cancer
Molecular Cancer Research, 2013Co-Authors: Hueiting Su, Chingchieh Weng, Pijung Hsiao, Lihua Chen, Yuwen Chen, Kuanghung ChengAbstract:The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-b1/Smad4 pathway in PDAC. We examined nestin expression after TGF-b1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-b1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-b1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxiainduced expression of nestin is mediated by the TGF-b1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-b1-induced EMT. Antinestin therapeutics may serve as a potential treatment for PDAC metastasis. Mol Cancer Res; 11(7); 768–79. � 2013 AACR.
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stem cell marker nestin is critical for tgf β1 mediated tumor progression in pancreatic cancer
Molecular Cancer Research, 2013Co-Authors: Chingchieh Weng, Pijung Hsiao, Lihua Chen, Yuwen Chen, Tzulei Kuo, Kungkai Kuo, Kuanghung ChengAbstract:The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-β1/Smad4 pathway in PDAC. We examined nestin expression after TGF-β1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-β1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-β1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-β1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-β1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.
Jing Zheng - One of the best experts on this subject based on the ideXlab platform.
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the r130s mutation significantly affects the function of pRestin the outer hair cell motor protein
Journal of Molecular Medicine, 2016Co-Authors: Satoe Takahashi, Jing Zheng, Mary Ann Cheatham, Kazuaki HommaAbstract:A missense mutation, R130S, was recently found in the pRestin gene, SLC26A5, of patients with moderate to severe hearing loss (DFNB61). In order to define the pathology of hearing loss associated with this missense mutation, a recombinant pRestin construct harboring the R130S mutation (R130S-pRestin) was generated, and its functional consequences examined in a heterologous expression system. We found that R130S-pRestin targets the plasma membrane but less efficiently compared to wild-type. The voltage operating point and voltage sensitivity of the motor function of R130S-pRestin were similar to wild-type pRestin. However, the motor activity of R130S-pRestin is greatly reduced at higher voltage stimulus frequencies, indicating a reduction in motor kinetics. Our study thus provides experimental evidence that supports a causal relationship between the R130S mutation in the pRestin gene and hearing loss found in patients with this missense mutation.
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pRestin dependence of outer hair cell survival and partial rescue of outer hair cell loss in pRestinv499g y501h knockin mice
PLOS ONE, 2015Co-Authors: Mary Ann Cheatham, Peter Dallos, Kazuaki Homma, Roxanne Edge, Emily L Leserman, Jing ZhengAbstract:A knockin (KI) mouse expressing mutated pRestinV499G/Y501H (499 pRestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 pRestin OHCs were normal in stiffness and longer than OHCs lacking pRestin, accelerated OHC death was unexpectedly observed relative to that documented in pRestin knockout (KO) mice. These observations imply an additional role of pRestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into pRestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 pRestin KI’s were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 pRestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 pRestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 pRestin and one copy of KO pRestin was also created to reduce quantities of 499 pRestin protein. Results show reduction in OHC death in chets, and in 499 pRestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 pRestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of pRestin’s motor function in cochlear amplification.
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the v499g y501h mutation impairs fast motor kinetics of pRestin and has significance for defining functional independence of individual pRestin subunits
Journal of Biological Chemistry, 2013Co-Authors: Kazuaki Homma, Jing Zheng, Chongwen Duan, Mary Ann Cheatham, Peter DallosAbstract:Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, pRestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which pRestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of pRestin provides vital information for unraveling the energy transduction mechanism of pRestin. We show that the V499G/Y501H mutation does not deprive pRestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H pRestin forms heteromers with wild-type pRestin and that the fast motor kinetics of wild-type pRestin is not affected by heteromer formation with V499G/Y501H pRestin. These results suggest that pRestin subunits are individually functional within a given multimer. Background: PRestin converts electrical energy into mechanical work. Results: The V499G/Y501H mutation significantly impairs fast motor kinetics of pRestin. Conclusion: Impaired kinetics is attributable to mutation at the Val-499 site that is conserved among SLC26 proteins regardless of their function as motors or transporters. Significance: V499G/Y501H mutated pRestin provides clues to the molecular mechanisms underlying somatic electromotility and thus cochlear amplification.
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interaction between the motor protein pRestin and the transporter protein vapa
Biochimica et Biophysica Acta, 2010Co-Authors: Soma Sengupta, Peter Dallos, Kazuaki Homma, Mary Ann Cheatham, Roxanne Edge, Katharine K Miller, Jing ZhengAbstract:PRestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify pRestin-associated proteins. Several proteins were recognized as potential pRestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and pRestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and pRestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from pRestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of pRestin. In other words, less VAPA protein is found in OHCs lacking pRestin. Thus, pRestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more pRestin protein appears at the plasma membrane when VAPA is co-expressed with pRestin. These data suggest that VAPA could be involved in pRestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.
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pRestin based outer hair cell electromotility in knockin mice does not appear to adjust the operating point of a cilia based amplifier
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Jiangang Gao, Jing Zheng, Mary Ann Cheatham, Xiang Wang, Sal Aguinaga, Kristin Huynh, Shuping Jia, Keiji Matsuda, Manish Patel, Peter DallosAbstract:The remarkable sensitivity and frequency selectivity of the mammalian cochlea is attributed to a unique amplification process that resides in outer hair cells (OHCs). Although the mammalian-specific somatic motility is considered a substrate of cochlear amplification, it has also been proposed that somatic motility in mammals simply acts as an operating-point adjustment for the ubiquitous stereocilia-based amplifier. To address this issue, we created a mouse model in which a mutation (C1) was introduced into the OHC motor protein pRestin, based on previous results in transfected cells. In C1/C1 knockin mice, localization of C1-pRestin, as well as the length and number of OHCs, were all normal. In OHCs isolated from C1/C1 mice, nonlinear capacitance and somatic motility were both shifted toward hyperpolarization, so that, compared with WT controls, the amplitude of cycle-by-cycle (alternating, or AC) somatic motility remained the same, but the unidirectional (DC) component reversed polarity near the OHC's presumed in vivo Resting membrane potential. No physiological defects in cochlear sensitivity or frequency selectivity were detected in C1/C1 or C1/+ mice. Hence, our results do not support the idea that OHC somatic motility adjusts the operating point of a stereocilia-based amplifier. However, they are consistent with the notion that the AC component of OHC somatic motility plays a dominant role in mammalian cochlear amplification.
Mary Ann Cheatham - One of the best experts on this subject based on the ideXlab platform.
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the r130s mutation significantly affects the function of pRestin the outer hair cell motor protein
Journal of Molecular Medicine, 2016Co-Authors: Satoe Takahashi, Jing Zheng, Mary Ann Cheatham, Kazuaki HommaAbstract:A missense mutation, R130S, was recently found in the pRestin gene, SLC26A5, of patients with moderate to severe hearing loss (DFNB61). In order to define the pathology of hearing loss associated with this missense mutation, a recombinant pRestin construct harboring the R130S mutation (R130S-pRestin) was generated, and its functional consequences examined in a heterologous expression system. We found that R130S-pRestin targets the plasma membrane but less efficiently compared to wild-type. The voltage operating point and voltage sensitivity of the motor function of R130S-pRestin were similar to wild-type pRestin. However, the motor activity of R130S-pRestin is greatly reduced at higher voltage stimulus frequencies, indicating a reduction in motor kinetics. Our study thus provides experimental evidence that supports a causal relationship between the R130S mutation in the pRestin gene and hearing loss found in patients with this missense mutation.
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pRestin dependence of outer hair cell survival and partial rescue of outer hair cell loss in pRestinv499g y501h knockin mice
PLOS ONE, 2015Co-Authors: Mary Ann Cheatham, Peter Dallos, Kazuaki Homma, Roxanne Edge, Emily L Leserman, Jing ZhengAbstract:A knockin (KI) mouse expressing mutated pRestinV499G/Y501H (499 pRestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 pRestin OHCs were normal in stiffness and longer than OHCs lacking pRestin, accelerated OHC death was unexpectedly observed relative to that documented in pRestin knockout (KO) mice. These observations imply an additional role of pRestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into pRestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 pRestin KI’s were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 pRestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 pRestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 pRestin and one copy of KO pRestin was also created to reduce quantities of 499 pRestin protein. Results show reduction in OHC death in chets, and in 499 pRestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 pRestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of pRestin’s motor function in cochlear amplification.
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the v499g y501h mutation impairs fast motor kinetics of pRestin and has significance for defining functional independence of individual pRestin subunits
Journal of Biological Chemistry, 2013Co-Authors: Kazuaki Homma, Jing Zheng, Chongwen Duan, Mary Ann Cheatham, Peter DallosAbstract:Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, pRestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which pRestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of pRestin provides vital information for unraveling the energy transduction mechanism of pRestin. We show that the V499G/Y501H mutation does not deprive pRestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H pRestin forms heteromers with wild-type pRestin and that the fast motor kinetics of wild-type pRestin is not affected by heteromer formation with V499G/Y501H pRestin. These results suggest that pRestin subunits are individually functional within a given multimer. Background: PRestin converts electrical energy into mechanical work. Results: The V499G/Y501H mutation significantly impairs fast motor kinetics of pRestin. Conclusion: Impaired kinetics is attributable to mutation at the Val-499 site that is conserved among SLC26 proteins regardless of their function as motors or transporters. Significance: V499G/Y501H mutated pRestin provides clues to the molecular mechanisms underlying somatic electromotility and thus cochlear amplification.
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interaction between the motor protein pRestin and the transporter protein vapa
Biochimica et Biophysica Acta, 2010Co-Authors: Soma Sengupta, Peter Dallos, Kazuaki Homma, Mary Ann Cheatham, Roxanne Edge, Katharine K Miller, Jing ZhengAbstract:PRestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify pRestin-associated proteins. Several proteins were recognized as potential pRestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and pRestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and pRestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from pRestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of pRestin. In other words, less VAPA protein is found in OHCs lacking pRestin. Thus, pRestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more pRestin protein appears at the plasma membrane when VAPA is co-expressed with pRestin. These data suggest that VAPA could be involved in pRestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.
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pRestin based outer hair cell electromotility in knockin mice does not appear to adjust the operating point of a cilia based amplifier
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Jiangang Gao, Jing Zheng, Mary Ann Cheatham, Xiang Wang, Sal Aguinaga, Kristin Huynh, Shuping Jia, Keiji Matsuda, Manish Patel, Peter DallosAbstract:The remarkable sensitivity and frequency selectivity of the mammalian cochlea is attributed to a unique amplification process that resides in outer hair cells (OHCs). Although the mammalian-specific somatic motility is considered a substrate of cochlear amplification, it has also been proposed that somatic motility in mammals simply acts as an operating-point adjustment for the ubiquitous stereocilia-based amplifier. To address this issue, we created a mouse model in which a mutation (C1) was introduced into the OHC motor protein pRestin, based on previous results in transfected cells. In C1/C1 knockin mice, localization of C1-pRestin, as well as the length and number of OHCs, were all normal. In OHCs isolated from C1/C1 mice, nonlinear capacitance and somatic motility were both shifted toward hyperpolarization, so that, compared with WT controls, the amplitude of cycle-by-cycle (alternating, or AC) somatic motility remained the same, but the unidirectional (DC) component reversed polarity near the OHC's presumed in vivo Resting membrane potential. No physiological defects in cochlear sensitivity or frequency selectivity were detected in C1/C1 or C1/+ mice. Hence, our results do not support the idea that OHC somatic motility adjusts the operating point of a stereocilia-based amplifier. However, they are consistent with the notion that the AC component of OHC somatic motility plays a dominant role in mammalian cochlear amplification.
