東華大學圖書館 |

Pathology of Primary Open Angle Glaucoma (POAG).

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Pathology of Primary Open Angle Glaucoma (POAG)./
作者:	He, Jingna.
面頁冊數:	1 online resource (200 pages)
附註:	Source: Dissertations Abstracts International, Volume: 81-09, Section: B.
Contained By:	Dissertations Abstracts International81-09B.
標題:	Ophthalmology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=27784078click for full text (PQDT)
ISBN:	9781392799093

Pathology of Primary Open Angle Glaucoma (POAG).
He, Jingna.

Pathology of Primary Open Angle Glaucoma (POAG). - 1 online resource (200 pages)

Source: Dissertations Abstracts International, Volume: 81-09, Section: B.

Thesis (Ph.D.)--The Chinese University of Hong Kong (Hong Kong), 2019.

Includes bibliographical references

Glaucoma, a leading cause of irreversible blindness worldwide, is a heterogeneous group of optic neuropathies characterized by cupping of the optic nerve head and visual field damage. Primary open-angle glaucoma (POAG) is the most common type of glaucoma. According to the highest intra-ocular pressure (IOP), POAG have been conventionally divided into high-tension POAG (HTG, IOP > 21 mmHg) and normal-tension POAG (NTG, IOP ≤ 21 mmHg).IOP is a major risk factor for POAG and lowering IOP is currently the only intervention that has been proven to be able to delay the progression of the disease. Trabecular meshwork / Schlemm's canal (TM / SC) conventional aqueous humor (AH) outflow pathway plays a key role in the regulation of IOP. These trabecular meshwork cells in the TM / SC outflow pathway are constantly subjected to oxidative stress attack generated from the physiological cellular metabolism. Oxidative stress represents increased intracellular reactive oxygen species (ROS) levels that could damage DNA, proteins and lipids. The lack of effective antioxidant mechanism in the TM may lead to cell decay, causing both morphological and functional alterations of the TM tissues. This scenario subsequently results in an increased AH outflow resistance and eventually IOP elevation. Autophagy is an essential and adaptive cellular survival mechanism to protect cells against various cellular stresses, including oxidative damages. These observations led to a hypothesis that inducing autophagy biochemically may relieve chronic oxidative stress in TM, and therefore alleviate the IOP elevation and the risk of having glaucoma. Currently IOP lowering agents mainly involve reducing the production of AH or promoting AH outflow through the unconventional intercellular space between ciliary muscle cells. These agents can only resolve the high IOP temporarily. On the other hand, as the TM / SC conventional AH outflow pathway controls more than 75% of the AH outflow, the knowledge about the autophagy in response to chronic oxidative stress in TM cells may help develop new therapeutic strategies to POAG. In this thesis, I aim to study the roles of autophagy in POAG. In the first part, I evaluated the roles of rapamycin in protecting TM cells from oxidative stress. In the second part, I screened for coding sequence variations in OPTN, an important gene in regulating autophagy, in Chinese NTG patients. In the third part, I further characterized the H486R OPTN mutation in TM cells.In this first part of this thesis, I induced chronic oxidative stress in human trabecular meshwork (TM-1) cells with 1 μM rotenone and investigated the levels of reactive oxygen species (ROS), autophagy and mitochondrial functions. Protective effects of rapamycin, an inducer of autophagy, were also investigated. Our data indicated that rotenone significantly increased oxidative stress, but not autophagy, in TM-1 cells. Rapamycin at 10 nM effectively suppressed the rotenone-induced cell apoptosis, as well as the ROS elevation. The protective effects of rapamycin could be associated to the induction of autophagy and removal of damaged mitochondria in TM-1 cells. Our results suggest autophagy has important roles in protecting TM-1 cells from oxidative stress, which could be further developed into a novel treatment to POAG.Optineurin (OPTN) is a receptor of autophagy. In the eye, optineurin is expressed in the TM, nonpigmented ciliary epithelium, and remarkably in the retina. The optineurin protein contains an ubiquitin-binding domain (UBD) (445-502 aa) and a LC3-interacting region (LIR) (169-184 aa), which allows optineurin to act like a bridge to bring ubiquitinated cargos to the autophagosome via LC3. In 2002, FIP-2 mutations were identified responsible for POAG in 54 families and this gene was renamed as "optineurin" (OPTN). Most of these families showed normal levels of IOP. Since then plenty of OPTN alternations were found in glaucoma patients. The subsequent studies did not show a statistical significant correlation of OPTN exclusively with HTG and there were variations in the frequency of coding and noncoding polymorphisms within OPTN in NTG among different ethnicity. So in the second of this thesis, I did OPTN coding region mutation screening in NTG among Chinese patients.Genomic DNA was extracted from 190 NTG patients and 201 control subjects. The thirteen (exon 4 - 16) exons of OPTN were amplified by polymerase chain reaction and analyzed by direct sequencing. Detected sequence changes were compared between NTG patients and control subjects. Seven sequence changes in OPTN were identified in both NTG patients and control subjects. Among them, c. 464G>A (T34T), c. 509C>T (T49T), c. 806G>A (V148V) and c. 959T>C (P199P) were synonymous codon changes, whilst c. 655T>A (M98K), c. 1996G>A (R545Q) and c. 1582T>C (I407T) were missense changes. Two previously reported heterozygous mutations, c.458G>A (E50K) in exon 4 and c.691_692insAG in exon 6, were not found in this study. Out of these seven OPTN sequence variants, c. 464G>A (T34T) was significantly associated with NTG in both the allelic and genotypic association analyses (allelic association: p = 0.0001, OR=2.20, 95% CI 1.46-3.31; genotypic association: p = 0.0001), whereas the association of other variants with NTG did not reach statistical significance (p>0.05). Variants c. 1582T>C (I407T) and c. 806G>A (V148V) were identified in one and two NTG patients, respectively, but not in control subjects.To further study the biological roles of these OPTN mutations, six sites on OPTN were mutated (E50K, M98K, S177A, S473A, H486R and S513A) to investigate their consequences in TM-1 cells under induced oxidative stress. Although some of these mutations were not found in our genetics study, they were still included for the functional investigations because of their important reported roles in autophagy and POAG. E50K, M98K and H486R were identified in POAG patients. S177A, S473A and S513A were loss-of-function mutations at the Tank-binding kinase 1 (TBK1) phosphorylation sites, which have been reported to cause defective autophagy. M98K was the missense codon change identified in this study. E50K showed a strong association with POAG, particularly with NTG. M98K and E50K are located in the TBK1 binding domain of OPTN (1 - 127 aa). H486R, located in the UBD of optineurin, was identified in POAG patient but not in control subjects. The oxidative stress levels, numbers of damaged mitochondria and apoptosis levels in the wild-type (WT) and mutant OPTN expressing TM-1 cells were investigated. Our results showed that H486R OPTN expressing cells had higher endogenous and induced oxidative stress levels than the WT OPTN expressing cells. More damaged mitochondria and higher apoptosis levels were also detected in H486R OPTN expressing cells. These results indicated H486R mutation in OPTN would affect the cellular functions of TM-1 cells. Further studies are needed to confirm the impacts of H486R OPTN in autophagy as this mutation site is situated in the UBD region, which is predicted to deliver the ubiquitinated cargos to the autophagosome. Our study could provide a preliminary understanding of the roles of OPTN H486R in regulating autophagy in TM cells, which suggest a role for the H486R OPTN mutation in POAG. These results could serve as a proof of concept to support the feasibility of this experimental approach in characterizing OPTN mutations. The same experimental approach could be used to study the other five variants.In conclusion, the defective optineurin function caused by H486R mutation would affect the cellular function of TM cells which may contribute to some of the pathologies of POAG. Also, the induction of autophagy level could protect TM cells from oxidative stress. Further exploring in the roles of autophagy may help develop new therapeutic strategies to POAG.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2023

Mode of access: World Wide Web

ISBN: 9781392799093Subjects--Topical Terms:

862704
Ophthalmology.
Subjects--Index Terms:

GlaucomaIndex Terms--Genre/Form:

542853
Electronic books.

Pathology of Primary Open Angle Glaucoma (POAG).
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520 $a Glaucoma, a leading cause of irreversible blindness worldwide, is a heterogeneous group of optic neuropathies characterized by cupping of the optic nerve head and visual field damage. Primary open-angle glaucoma (POAG) is the most common type of glaucoma. According to the highest intra-ocular pressure (IOP), POAG have been conventionally divided into high-tension POAG (HTG, IOP > 21 mmHg) and normal-tension POAG (NTG, IOP ≤ 21 mmHg).IOP is a major risk factor for POAG and lowering IOP is currently the only intervention that has been proven to be able to delay the progression of the disease. Trabecular meshwork / Schlemm's canal (TM / SC) conventional aqueous humor (AH) outflow pathway plays a key role in the regulation of IOP. These trabecular meshwork cells in the TM / SC outflow pathway are constantly subjected to oxidative stress attack generated from the physiological cellular metabolism. Oxidative stress represents increased intracellular reactive oxygen species (ROS) levels that could damage DNA, proteins and lipids. The lack of effective antioxidant mechanism in the TM may lead to cell decay, causing both morphological and functional alterations of the TM tissues. This scenario subsequently results in an increased AH outflow resistance and eventually IOP elevation. Autophagy is an essential and adaptive cellular survival mechanism to protect cells against various cellular stresses, including oxidative damages. These observations led to a hypothesis that inducing autophagy biochemically may relieve chronic oxidative stress in TM, and therefore alleviate the IOP elevation and the risk of having glaucoma. Currently IOP lowering agents mainly involve reducing the production of AH or promoting AH outflow through the unconventional intercellular space between ciliary muscle cells. These agents can only resolve the high IOP temporarily. On the other hand, as the TM / SC conventional AH outflow pathway controls more than 75% of the AH outflow, the knowledge about the autophagy in response to chronic oxidative stress in TM cells may help develop new therapeutic strategies to POAG. In this thesis, I aim to study the roles of autophagy in POAG. In the first part, I evaluated the roles of rapamycin in protecting TM cells from oxidative stress. In the second part, I screened for coding sequence variations in OPTN, an important gene in regulating autophagy, in Chinese NTG patients. In the third part, I further characterized the H486R OPTN mutation in TM cells.In this first part of this thesis, I induced chronic oxidative stress in human trabecular meshwork (TM-1) cells with 1 μM rotenone and investigated the levels of reactive oxygen species (ROS), autophagy and mitochondrial functions. Protective effects of rapamycin, an inducer of autophagy, were also investigated. Our data indicated that rotenone significantly increased oxidative stress, but not autophagy, in TM-1 cells. Rapamycin at 10 nM effectively suppressed the rotenone-induced cell apoptosis, as well as the ROS elevation. The protective effects of rapamycin could be associated to the induction of autophagy and removal of damaged mitochondria in TM-1 cells. Our results suggest autophagy has important roles in protecting TM-1 cells from oxidative stress, which could be further developed into a novel treatment to POAG.Optineurin (OPTN) is a receptor of autophagy. In the eye, optineurin is expressed in the TM, nonpigmented ciliary epithelium, and remarkably in the retina. The optineurin protein contains an ubiquitin-binding domain (UBD) (445-502 aa) and a LC3-interacting region (LIR) (169-184 aa), which allows optineurin to act like a bridge to bring ubiquitinated cargos to the autophagosome via LC3. In 2002, FIP-2 mutations were identified responsible for POAG in 54 families and this gene was renamed as "optineurin" (OPTN). Most of these families showed normal levels of IOP. Since then plenty of OPTN alternations were found in glaucoma patients. The subsequent studies did not show a statistical significant correlation of OPTN exclusively with HTG and there were variations in the frequency of coding and noncoding polymorphisms within OPTN in NTG among different ethnicity. So in the second of this thesis, I did OPTN coding region mutation screening in NTG among Chinese patients.Genomic DNA was extracted from 190 NTG patients and 201 control subjects. The thirteen (exon 4 - 16) exons of OPTN were amplified by polymerase chain reaction and analyzed by direct sequencing. Detected sequence changes were compared between NTG patients and control subjects. Seven sequence changes in OPTN were identified in both NTG patients and control subjects. Among them, c. 464G>A (T34T), c. 509C>T (T49T), c. 806G>A (V148V) and c. 959T>C (P199P) were synonymous codon changes, whilst c. 655T>A (M98K), c. 1996G>A (R545Q) and c. 1582T>C (I407T) were missense changes. Two previously reported heterozygous mutations, c.458G>A (E50K) in exon 4 and c.691_692insAG in exon 6, were not found in this study. Out of these seven OPTN sequence variants, c. 464G>A (T34T) was significantly associated with NTG in both the allelic and genotypic association analyses (allelic association: p = 0.0001, OR=2.20, 95% CI 1.46-3.31; genotypic association: p = 0.0001), whereas the association of other variants with NTG did not reach statistical significance (p>0.05). Variants c. 1582T>C (I407T) and c. 806G>A (V148V) were identified in one and two NTG patients, respectively, but not in control subjects.To further study the biological roles of these OPTN mutations, six sites on OPTN were mutated (E50K, M98K, S177A, S473A, H486R and S513A) to investigate their consequences in TM-1 cells under induced oxidative stress. Although some of these mutations were not found in our genetics study, they were still included for the functional investigations because of their important reported roles in autophagy and POAG. E50K, M98K and H486R were identified in POAG patients. S177A, S473A and S513A were loss-of-function mutations at the Tank-binding kinase 1 (TBK1) phosphorylation sites, which have been reported to cause defective autophagy. M98K was the missense codon change identified in this study. E50K showed a strong association with POAG, particularly with NTG. M98K and E50K are located in the TBK1 binding domain of OPTN (1 - 127 aa). H486R, located in the UBD of optineurin, was identified in POAG patient but not in control subjects. The oxidative stress levels, numbers of damaged mitochondria and apoptosis levels in the wild-type (WT) and mutant OPTN expressing TM-1 cells were investigated. Our results showed that H486R OPTN expressing cells had higher endogenous and induced oxidative stress levels than the WT OPTN expressing cells. More damaged mitochondria and higher apoptosis levels were also detected in H486R OPTN expressing cells. These results indicated H486R mutation in OPTN would affect the cellular functions of TM-1 cells. Further studies are needed to confirm the impacts of H486R OPTN in autophagy as this mutation site is situated in the UBD region, which is predicted to deliver the ubiquitinated cargos to the autophagosome. Our study could provide a preliminary understanding of the roles of OPTN H486R in regulating autophagy in TM cells, which suggest a role for the H486R OPTN mutation in POAG. These results could serve as a proof of concept to support the feasibility of this experimental approach in characterizing OPTN mutations. The same experimental approach could be used to study the other five variants.In conclusion, the defective optineurin function caused by H486R mutation would affect the cellular function of TM cells which may contribute to some of the pathologies of POAG. Also, the induction of autophagy level could protect TM cells from oxidative stress. Further exploring in the roles of autophagy may help develop new therapeutic strategies to POAG.
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538 $a Mode of access: World Wide Web
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653 $a Glaucoma
653 $a Primary open angle glaucoma
653 $a Intra-ocular pressure
655 7 $a Electronic books. $2 lcsh $3 542853
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710 2 $a The Chinese University of Hong Kong (Hong Kong). $3 1017547
773 0 $t Dissertations Abstracts International $g 81-09B.
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=27784078 $z click for full text (PQDT)