Exfoliation Syndrome: A Disease of Autophagy and LOXL1 Proteopathy

Abstract

Exfoliation syndrome (XFS) is an age-related disease involving the deposition of aggregated fibrillar material (exfoliation material) at extracellular matrices in tissues that synthesize elastic fibers. Its main morbidity is in the eye, where exfoliation material accumulations form on the surface of the ciliary body, iris, and lens. Exfoliation glaucoma (XFG) occurs in a high proportion of persons with XFS and can be a rapidly progressing disease. Worldwide, XFG accounts for about 25% of open-angle glaucoma cases. XFS and XFG show a sharp age-dependence, similarly to the many age-related diseases classified as aggregopathies. Progress in understanding the cellular bases for XFS/XFG has been slowed by a lack of experimental models. Working with primary human tenon fibroblasts (TF) derived from trabeculectomies of XFG patients and age-matched primary open-glaucoma controls, we found that TF from XFG cells display many of the functional features observed in cells from other protein aggregate diseases, such as Parkinson, Alzheimer, Huntington, and age-related macular degeneration. We have documented defects in lysosomal positioning, microtubule organization, autophagy processing rate, and mitochondrial health. In regard to failure of lysosomal and autophagosome positioning in XFG cells, we have found that XFG TF are unable to establish the transnuclear microtubule organizing center that is required for efficient centripetal vesicular locomotion along microtubules. In regard to potential sources of the autophagy malfunction, we have directed our attention to a potential role of the lysyl oxidase-like 1 protein (LOXL1), the elastic fiber catalyst that displays variant-dependent association with risk for XFG. Our experiments show that (a) in XFG cells, a substantial fraction of LOXL1 is processed for degradation by the autophagic system; (b) most of the LOXL1 N-terminus domain exists in a highly disordered state, a condition known to greatly increase the frequency of polypeptide misfolding; (c) that maximum misfolding occurs at amino acid position 153, the location of the high risk variant G153D; and (d) that replacement of glycine (G) by aspartate (D) there results in a substantial decrease in disorder within the 20 amino acid surrounding domain. Finally, we show that clusterin, a protein that can be induced by the presence of intracellular, or extracellular aggregates, is uniformly overexpressed in XFG TF. The implications of our results for a theory relating XFG to cellular aggregopathy are discussed.

Figure 1. The ERAD and MAPS pathways

Misfolded proteins in the lumen of the ER activate the unfolded protein response (UPR) in which translation of some proteins is halted, while protein expression of chaperones is increased. If this response does not achieve refolding of the protein to a correct conformation, then the misfolded protein is retrotranslocated out of the ER, through the endoplasmic reticulum-associated degradation (ERAD) pathway. In the cytosol, ubiquitin ligases ubiquitinate the misfolded protein targeting it for degradation in the proteasome or the polypeptides are packed into aggresomes (described in Figure 2). Recently, it has been reported that when the the proteasome is dysfuctional, as occurs in many age-related diseases, misfolded proteins maybe disposed by secretion through a novel misfolding-associated protein secretion (MAPS) pathway. An isoform of the deubiquitinase, USP19 localized at the ER outer membrane plays a key role in MAPS.

Figure 2. Macroautophagy

Macroautophagy involves biogenesis of autophagosomes from precursor phagophores within the cytosol. Attachment to microtubules is orchestrated by the lipidated form of LC3 (LC3-II). Autophagasomes engulf ‘macroscopic’ cellular detritus, naturally decaying organelles, in particular mitochondria (i.e., mitophagy) and misfolded or denatured proteins which have been ‘packed’ into particulates called aggresomes. These processess occur simultaneously with centripetal dyenin-dependent traffic of these organelles along microtubules towards the juxtanuclear microtubule organizing center (MTOC). The latter is formed by the anchoring of microtubules (composed of α and β tubulin) to a matching ‘ring’ made of γ-tubulin. This ring is held together by several other associated proteins to form a conical structure known as the γ-tubulin ring complex (γ-TuRC; ). γ-TuRCs, in turn, are densely packed by attachment to the cell centrosomes ( ) via the adaptor protein ninein (NIN; ). Magnified MTOC detail: The insert depicts magnified details of the fusion events in the MTOC proximity. Early and late endosomes can fuse directly with lysosomes or with autophagasomes to form an amphisome. Both autophagosomes and amphisomes then fuse with the lysosome creating the autolysosome. In this transient structure the vesicular detritus cargo are lytically degraded into basic biochemical building blocks by the lysosome’s acidic hydrolases. Dense organelle accumulation at the MTOC greatly enhances these intervesicular fusions. Detritus that resists the sequential processing described above may accumulate in body inclusions (e.g., Lewy body in senile dementia), from where it may be exported. Re-uptake of these aggregates by the same (phagocytosis) will augment the load of undegredable protein in repetitive cycles. Furthermore, if exocytosis of aggregates or misfolded protein by the MAPS pathway described in Figure 1) is operative then the aggregopathy may propagate to adjacenct cells by particulate phagocytosis or endocytosis, as demonstrated for tau protein propagation in Alzheimer’s and α-synuclein protein in Parkinson’s.

Figure 3. Salient phenotypic differences between TF derived from XFG and POAG donors

XFG and POAG TF were seeded under serum-free (starvation) conditions. A,B. Distribution of microtubules (β-tubulin, green) and lysosomes (LAMP1, pink), Bar=50um. C,D Magnified images. In XFG cells, with the exception of rare cells, the β-tubulin stain remains outside the nucleus and the lysosomes are minimally condensed. In the POAG cells lysosomes and β-tubulin staining is highly concentrated at a typical MTOC structure. E,F. In XFG cells the concentration of γ-tubulin remains outside the nucleus and is either small of amorphous γ-tubulin staining (arrow). In POAG cells, a large fraction of the stain is localized in a tubular or conical small structure consistent with the γ-TurC morphology that clearly penetrates deep into the nucleus (arrow). Bar = 25um. N=3.

Figure 4. Effect of Bafilomycin-A1 and Spautin-1 on LOXL1 protein

XFS and POAG TF were seeded in serum-free (starvation) conditions for 18 hours with 0.5uM Bafilomycin-A1 (BafA), 10uM Spautin-1 (Sp) or in control medium, without inhibitors. Western blot for LOXL1 shows that in XFG cells the amount of LOXL1 increases after treatment with autophagy inhibitors, whereas in POAG cells these inhibitors reduce LOXL1 amounts. Loading control, GAPDH. Statistical results for N = 4 are shown as the percent change (up or down) for XFG and POAG cells with each inhibitor.

Figure 5. Clusterin cellular distribution in 3D culture and protein expression in 2D culture

A-D) XFS and POAG TF were cultured in 1% FBS containing media with vitamin C for 1 month creating 3D self-synthesizing constructs. Cells were fixed and immunostained for clusterin. A,B bar = 50um. C,D Magnified images. N=3. In XFG cells, when compared with POAG cells, clusterin is in small globular structures and aligns with linear fibrillar arrays (arrow). E) Western blots of XFS and POAG cells cultured in starvation medium for two days. Loading control, GAPDH N=4. F) Quantification of Western blot.

Figure 6. Structural disorder in LOXL1

A. LOXL1 contains multiple disordered domains on its N-terminus. Particularly significant is a wide peak of disorder in the 151-170 amino acid domain. It includes the highest risk allele for XFG where homozygosity for glycine (G) at position 153 is associated with 98% of the XFG cases in a broad ethnic diversity USA population 56. The site of copper binding (site of deaminase activity) is indicated. B. Replacement of glycine by aspartic acid (D) at pos. 153 results in a ~ 8-10 % decrease in disorder probability in the 151-180 residue span. C. Comparison of disorder probability for the LOX family of proteins shows domains with high probability disorder exist only in the much smaller LOX protein; LOXL2-4 appear to be highly structured proteins. All sequences have been aligned using a preserved domain of very low disorder probability present at the C-terminus (blue line).