Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells

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.</p

T-RFLP shows greater similarity based on sampling technique than colonic region within individuals.

<p>A. Electropherograms of T-RFLP sequence profiles generated from cecal and rectal biopsies from sample 1, superimposed on each other, illustrating close approximation of sample profiles. B. Electropherograms of T-RFLP sequence profiles generated from cecal and rectal brushings from sample 1. C. Mean Bray-Curtis Similarity Index (BCI) values between cecal and rectal biopsy samples (0.77 (0.73–0.81)), cecal and rectal brush samples (0.62 (0.57–0.77)) and biopsy and brush samples at cecum (0.35 (0.27–0.47)) and rectum (0.46 (0.44–0.49)).</p

Brush sampling retrieves an enriched bacterial sample with less host eukaryotic DNA.

<p>A. Agarose gel with bands representing human GAPDH gene, illustrating the reduced quantity of human DNA in brush samples compared to biopsy samples. B. Agarose gel with bands representing the 16S rRNA pan-bacterial gene, illustrating the increased proportion bacterial DNA which is sampled by brushing compared to biopsy sampling.</p

Reduced diversity in luminal brush samples when compared with mucosal biopsies.

<p>A. Boxplot of Shannon Diversity Index values for brush and biopsy samples (median value for biopsy samples 3.36 (3.3–3.5), median value for brush samples 2.94 (2.8–3.1) (<i>P</i><.001). Median values for the working channel 2.6 (2.5–2.8). B. Boxplot of TRF abundance (median value for biopsy samples 75 (63–81), median value for brush samples 44 (36–52), (<i>P</i><.001)). Median value for the working channel 29 (27–32). C. Boxplot of TRF evenness (median value for biopsy samples 0.81 (0.78–0.82), median value for brush samples 0.8 (0.75–0.83) (<i>P</i> = 0.591). Median values for the working channel 0.78 (0.76–0.8).</p

Separate clustering of luminal brush and mucosal biopsy samples between individuals.

<p>A. Non-metric Multidimensional Scaling (NMDS) reveals samples do not cluster based on colonic region (black = cecum; red = rectum). B. NMDS analysis reveals that samples cluster according to their cross-sectional location with respect to the host bacterial interface (black = Mucosal biopsy; red = luminal brush).</p

Poor discrimination between individuals by samples derived from colonoscope channel.

<p>A. NMDS plot (stress 0.085) highlighting separation of biopsy samples (black) from brush samples and samples taken from the working channel of the colonoscope (red and green, respectively). B. Neighbor-joining dendrogram of the same samples in A, illustrating that samples derived from the working channel do not discriminate as accurately between individuals as brush samples.</p

Characteristics of study volunteers.

<p>Characteristics of study volunteers.</p