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Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells.

McElnea EM, Hughes E, McGoldrick A, McCann A, Quill B, Docherty N, Irnaten M, Farrell M, Clark AF, O'Brien CJ, Wallace DM - BMC Ophthalmol (2014)

Bottom Line: Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot.A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells.Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein.

View Article: PubMed Central - PubMed

Affiliation: University College Dublin School of Medicine and Health Sciences, University College Dublin, Dublin, Ireland. mcelneaelizabeth@gmail.com.

ABSTRACT

Background: Disease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell's constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes.

Methods: The number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed.

Results: A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors.

Conclusions: Increased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.

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LC cell auto-fluorescence reflecting cellular lipopfuscin content. The lipofuscin auto-fluorescence emitted by approximately 10,000 cells in the yellow-green range of the spectrum (563-607 nm, FL-1 channel) was quantified by flow cytometry. Glaucomatous LC cells demonstrated increased whole cell auto-fluorescence relative to normal LC cells (83.6 ± 45.1 MFI vs. 41.0 ± 3.9 MFI, p = 0.02, n = 3).
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Fig2: LC cell auto-fluorescence reflecting cellular lipopfuscin content. The lipofuscin auto-fluorescence emitted by approximately 10,000 cells in the yellow-green range of the spectrum (563-607 nm, FL-1 channel) was quantified by flow cytometry. Glaucomatous LC cells demonstrated increased whole cell auto-fluorescence relative to normal LC cells (83.6 ± 45.1 MFI vs. 41.0 ± 3.9 MFI, p = 0.02, n = 3).

Mentions: Intracellular auto-fluorescent material was found under the FITC filter at live cell fluorescence microscopy in LC cells from both normal and glaucoma donors. As shown in Figure 2, whole cell auto-fluorescence, reflective of cellular lipofuscin content [32] and measured at the 563-607 nm wavelength band, was increased in GLC [auto-fluorescence 83.6 ± 45.1 mean fluorescence intensity (MFI)] compared to NLC (auto-fluorescence 41.0 ± 3.9 MFI, p = 0.02, n = 3) cell groups. The data from Figures 1B, C and 2 has been summarized in Table 1.Figure 2


Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells.

McElnea EM, Hughes E, McGoldrick A, McCann A, Quill B, Docherty N, Irnaten M, Farrell M, Clark AF, O'Brien CJ, Wallace DM - BMC Ophthalmol (2014)

LC cell auto-fluorescence reflecting cellular lipopfuscin content. The lipofuscin auto-fluorescence emitted by approximately 10,000 cells in the yellow-green range of the spectrum (563-607 nm, FL-1 channel) was quantified by flow cytometry. Glaucomatous LC cells demonstrated increased whole cell auto-fluorescence relative to normal LC cells (83.6 ± 45.1 MFI vs. 41.0 ± 3.9 MFI, p = 0.02, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265474&req=5

Fig2: LC cell auto-fluorescence reflecting cellular lipopfuscin content. The lipofuscin auto-fluorescence emitted by approximately 10,000 cells in the yellow-green range of the spectrum (563-607 nm, FL-1 channel) was quantified by flow cytometry. Glaucomatous LC cells demonstrated increased whole cell auto-fluorescence relative to normal LC cells (83.6 ± 45.1 MFI vs. 41.0 ± 3.9 MFI, p = 0.02, n = 3).
Mentions: Intracellular auto-fluorescent material was found under the FITC filter at live cell fluorescence microscopy in LC cells from both normal and glaucoma donors. As shown in Figure 2, whole cell auto-fluorescence, reflective of cellular lipofuscin content [32] and measured at the 563-607 nm wavelength band, was increased in GLC [auto-fluorescence 83.6 ± 45.1 mean fluorescence intensity (MFI)] compared to NLC (auto-fluorescence 41.0 ± 3.9 MFI, p = 0.02, n = 3) cell groups. The data from Figures 1B, C and 2 has been summarized in Table 1.Figure 2

Bottom Line: Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot.A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells.Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein.

View Article: PubMed Central - PubMed

Affiliation: University College Dublin School of Medicine and Health Sciences, University College Dublin, Dublin, Ireland. mcelneaelizabeth@gmail.com.

ABSTRACT

Background: Disease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell's constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes.

Methods: The number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed.

Results: A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors.

Conclusions: Increased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.

Show MeSH
Related in: MedlinePlus