Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

remains largely unknown. Genomic approaches to understanding glaucoma etiology are further complicated by limited knowledge of gene regulation, with genetic detection of defective promoter regions and abnormally expressed proteins of particular challenge. Classical proteomic comparison of diseased and normal tissues have proven useful for deciphering a variety of pathological mechanisms, and recent reports support the utility of proteomic approaches for obtaining insights into POAG pathology. This review focuses on proteomic analyses which have identified abnormal expressed proteins in glaucomatous trabecular meshwork (TM) (9–11) and optic nerve (12). These studies have implicated a role for (i) cochlin in the TM in impairing aqueous humor outflow and elevating IOP and (ii) peptidyl arginyl deiminase 2 in the optic nerve through citrullination and structural disruption of proteins, including those associated with myelin.

COCHLIN

Cochlin Structure, Function, and Disease Involvement

Cochlin, the product of the COCH (Coagulation factor C Homology) gene located on human chromosome 14q12-q13, is a secreted protein comprising the major non-collagen component of the extracellular matrix (ECM) of the inner ear (13,14). The function of cochlin is unknown; however, cochlin deposits in the cochlea and COCH mutations have been linked with DFNA9, an autosomal dominant non-syndromic auditory and vestibular disorder (13,15). Mutations in COCH have also been associated with Ménière’s disease (with hearing loss and vertigo) and presbyacusis (age-related hearing loss) (13,15). Cochlin is a secreted glycoprotein with three apparent isoforms with masses of about 40 KDa, 46 KDa, and 60 KDa (13,16). A short predicted signal peptide is encoded by COCH along with an N-terminal factor C homology (FCH) domain and two von Willebrand factor A-like (vWFA) domains. The FCH domain is named after the coagulation factor from horseshoe crabs (Limulus factor C) that initiates a host defense coagulation cascade and is also referred as the LCCL domain, named after Limulus factor C, cochlear protein (Coch 5b2), and the late gestation lung protein (Lgl1). The FCH domain is common to several other proteins where it appears to be involved in antibody-independent host defense (17). DFNA9-causing mutations in the FCH domain of cochlin are thought to interfere with proper protein folding (13,18). vWFA domains are found in other proteins such as cell adhesion and ECM proteins and integrin receptors that form multiprotein complexes (19). The vWFA and FCH domains both appear likely to play a role in cochlin interactions with other proteins.

Proteomic Detection of Cochlin in Glaucomatous TM

Cochlin was initially associated with POAG by a classical proteomic comparison of glaucomatous and normal TM. Human POAG TM tissues were obtained from trabeculectomies performed at the Cole Eye Institute, Cleveland Clinic Foundation, and normal, control TM was from clinically documented cadaver eyes from the Cleveland Eye Bank (9). TM protein extracts were fractionated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), gel slices were excised, and proteins were identified using liquid chromatography tandem mass spectrometry (LC MS/MS) and bioinformatic methods (9). A total of 368 proteins were identified,

52 of which were detected only in glaucomatous TM. Cochlin was the most frequently identified protein from glaucomatous TM and was not detected in control TM (see Table 1). Subsequent western analysis confirmed the presence of cochlin in TM from 20 POAG donors and its absence in TM from 20 normal, control donors (9). The significance of other proteins detected only in human glaucomatous TM remains open for further study.

Cochlin expression in TM was compared by western analysis in tissues from the glaucomatous DBA/2J mouse (20–22) and from non-glaucomatous mouse strains BALBc/ByJ, CD1, and C57BL/6J. The DBA/2J mouse line exhibits increased IOP at 6–8 months of age, with progressive damage to the optic nerve and progressive hearing loss. Cochlin was detected in DBA/2J TM as early as 2–3 weeks of age and progressively increased cochlin expression was observed at 3, 10, and 16 weeks of age. In comparison, cochlin was not detected in TM from age-matched C57BL/6J, CD1, or BALBc/ByJ mice that do not develop increased IOP (23).

Table 1

Select Proteins Identified Only in Glaucomatous Trabecular Meshwork (TM)

Select proteins identified by LC MS/MS of human trabecular meshwork from six normal and six primary open angle glaucoma (POAG) tissue donors (9). Also shown is the number of glaucomatous TM donors from which the indicated protein was detected. Swiss-Protein database accession numbers are shown in plain font and NCBI accession number in italics.

Cochlin Deposits in Glaucomatous TM

Microfibrillar deposits and sheath-derived plaques have been reported in the TM of POAG donors (24,25). Histochemical analyses of human TM revealed that cochlin, in association with mucopolysaccharide, forms acidophilic deposits within glaucomatous TM but not in control TM (9). The observed regions of cochlin deposits appeared around Schlemm’s canal and were found to be acellular (see Fig. 1). Comparable histochemical analyses of 8-month old mice also revealed cochlin deposits in DBA/2J TM but not in C57BL/6J TM.

Cochlin Versus Collagen II in Glaucomatous TM

Quantitative western analysis was used to compare expression levels of cochlin and type II collagen in the TM. An age-dependent increase in cochlin content was observed in human glaucomatous TM, with a concomitant decrease in collagen II, while normal human TM exhibited no significant change in collagen II with age (9). Type II collagen expression was also examined in DBA/2J and C57BL/6J mice. Similar to the human condition, glaucomatous DBA/2J mice exhibit a progressive, age-dependent decline in type II collagen in the TM (see Fig. 2). This change is not a feature of normal murine aging, as type II collagen levels remain stable in older C57BL/6J mice. These observations suggest that cochlin may disrupt the TM architecture, rendering extracellular matrix (ECM) components like collagen more susceptible to degradation.

Fig. 1. Histochemical localization of cochlin in human trabecular meshwork (TM). Panels A and B show representative immunohistochemical analysis with chicken polyclonal anti-cochlin, rhodamine-conjugated secondary antibody, and immunofluorescence detection: (A) Normal TM from a 75-year-old Caucasian, female donor; (B) primary open angle glaucoma (POAG) TM from a 77-year-old Caucasian, female donor. Arrows indicate cochlin deposits in panel B that are approximately 80–150 μm. Asterisk indicates Schlemm’s canal. Panels C and D show representative light microscopy with Movat’s pentachrome-stained TM. (C) Normal TM from a 75-year-old Caucasian female; (D) POAG TM from a 77-year-old Caucasian female. Arrow indicates MPS deposit around Schlemm’s canal. (Bars represent 40 μm in A, B and 100 μm in C, D.) [Reproduced from Bhattacharya et al. (9) with permission].

that cochlin may undergoing multimeric aggregation in response to IOP fluctuations and different aqueous humor flow regimes, and thus initiate deposit formation in the TM. We further hypothesize that this may also contribute to collagen degradation. Progressively increased levels of cochlin in the TM, where it is not normally expressed, may change the manner in which fibrillar collagens interact, disrupting the integrity of the TM and triggering proteolytic degradation, collapse, and debris deposition that obstructs aqueous outflow (28,29). Future research is needed to determine whether cochlin expression and deposition in the TM is a cause of increased IOP or a secondary effect.

Future Cochlin Studies

Current results suggest that cochlin contributes to elevated IOP in POAG through altered interactions within the TM involving cell aggregation, mucopolysaccharide/ protein deposition, and obstruction of the aqueous humor circulation. Toward a better understanding of a possible pathophysiological role for cochlin in POAG, future studies would be useful that characterize cochlin expression in other animal models exhibiting elevated IOP (30–32). Likewise, targeted overexpression of cochlin in the TM of non-glaucomatous mice and cochlin knockdown experiments in glaucomatous animals would be informative with regard to IOP. Another approach would be the development of a DBA/2J-like mouse lacking cochlin by crossing a cochlin knockout allele (33) onto the DBA/2J background. If cochlin expression in the TM plays a critical role in generating elevated IOP and glaucomatous damage, then the absence of cochlin should modify the pathophysiology.

PEPTIDYL ARGININE DEIMINASE 2

Peptidyl Arginine Deiminase 2 Structure, Function, and Disease

Involvement

Peptidyl arginine deiminase 2 (PAD2, also known as protein-arginine deiminase type II) is the 75.5 kDa product of the PADI2 gene located on human chromosome 1p35.2-p35.1. PAD2 converts protein arginine to citrulline and its enzyme activity is modulated by calcium (34,35). Four other peptidyl arginine deiminase isoforms have been detected in humans, including PAD1, 3, 4, and 6, which like PAD2, exhibit cytosolic expression (34,36). PAD4 has also been found in the nucleus where it converts histone arginine to citrulline, impacting transcriptional regulation of gene expression by modulating arginine methylation (37,38). Rodent PAD2 has been detected in muscle, uterus, salivary gland, pancreas, spinal cord, and brain (39–42), however, PAD isoforms have been identified in a variety of other tissues, including hair follicles, epidermis, prostate, testis, ovary, placenta, spleen, thymus, eosinophiles, neutrophiles, and peripheral leukocytes (40,43). The unprocessed PAD isoforms range in size from 663 to 686 amino acids ( 74–77 kDa) and are typically composed of three domains, an N-terminal non-catalytic domain of 110 amino acids, a central region of 160 amino acids, and a larger C-terminal catalytic domain of 385 residues. PADs have been implicated in demyelinating diseases (39), and citrullination has been implicated in diseases such as autoimmune rheumatoid arthritis (44–46), multiple

methylation in POAG optic nerve as in histones (37,38) or increased demethylimination (37) reduces arginine methylation in optic nerve is not yet known. However, increased levels of PAD2 in POAG optic nerve correlate with increased citrullination and significantly altered posttranslational modifications that would change the charge properties of arginine-containing proteins.

Mass spectrometry, western analysis, and immunoprecipitation were used to identify citrinullated proteins in optic nerve extracts. Myelin basic protein (MBP) was identified as a major citrullinated protein in POAG optic nerve, and more citrullinated MBP was detected in POAG than in normal optic nerve extracts (see Fig. 4). Other proteins identified among the anti-citrulline immunoprecipitation products from POAG optic nerve included myelin proteolipid protein, myelin associated glycoprotein, myelin P0 protein, and myelin oligodendrocyte protein (see Table 3).

Factors Influencing PAD2 Expression

A variety of factors trigger PAD2 expression. We explored the effect of pressure and calcium on PAD2 expression in primary rat cortex astrocyte cultures (12). Shortterm elevated pressure generated increased PAD2 expression and citrullination for several days following restoration of atmospheric pressure (12). High levels of PAD2 were also detected in optic nerve extract from a POAG donor with elevated IOP

Fig. 4. Immunoprecipitation (IP) of citrullinated proteins in human optic nerve. (A) Coomassie blue detection of IP products from optic nerve extracts from human primary open angle glaucoma (POAG) (G) and normal (N) tissues with anti-citrulline or anti-myelin basic protein (MBP) and of POAG and normal optic nerve extracts without IP (15 μg). (B) Western detection with anti-citrulline of IP products from anti-citrulline or anti-MBP recovered from POAG and normal optic nerve extracts. (C) Western detection with anti-MBP of IP products from anti-citrulline or anti-MBP recovered from POAG and normal optic nerve extracts.

(D) Western detection with antibodies to myelin proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and MBP of anti-citrulline IP products from glaucomatous optic nerve [Reproduced from Bhattacharya et al. (12) with permission].

Select proteins identified from anti-citrulline immunprecipitation products from primary open angle glaucoma (POAG) optic nerve (12). Peptide matches indicate the number of peptide identifed by LC MS/MS sequence analsyis. Swiss-Protein database accession numbers are shown.

but no previous pharmacological or surgical intervention. In other POAG optic nerve extracts, PAD2 remained elevated after trabeculectomy with or without treatment with a calcium modulator (verapamil) and after the IOP returned to normal levels. Astrocyte cultures subjected to elevated pressure also exhibited increased intracellular calcium, concomitant with elevated level of PAD2 and citrullination while cultures treated with a calcium chelator exhibited decreased PAD2 (34,35). Increased IOP in glaucoma has been associated with increased intracellular calcium (51), which may elicit the observed increase in PAD2 activity and citrullination in the optic nerve (12). In vivo results from POAG donors and the DBA/2J glaucomatous mouse model support elevated IOP as a possible contributing factor to PAD2 expression.

Northern analysis of PAD2 mRNA levels in astrocytes and in human optic nerve extracts was used to determine whether increased PAD2 mRNA was associated with

increased protein expression. The amount of the PAD2 transcript was found to be very similar between pressure treated or untreated astrocytes and between normal and POAG optic nerve extracts, suggesting that overexpression of PAD2 is stimulated by post transcriptional mechanisms. Other evidence supporting translational modulation of PAD2 expression was obtained from normal and POAG optic nerve extracts depleted of PAD2 and polyA RNA. Addition of exogenous polyA RNA to the depleted extracts resulted in a large increase in PAD2 expression in the POAG optic nerve extracts but not in the normal, control extracts.

Although increased PAD2 transcripts are not required for increased PAD2 protein, decreasing PAD2 mRNA with short hairpin RNA (shRNA) appears to be a promising approach to reducing pressure-induced PAD2 and citrullination. Immunohistochemical and western analyses showed that pressure-treated astrocytes transfected with a PAD2 shRNA (but not with a random control shRNA) exhibited both reduced PAD2 expression and reduced citrullination, with no apparent changes in cell morphology.

Possible Roles for PAD2 in Glaucoma Pathology

Multiple citrullinated myelin-associated proteins were detected in POAG optic nerve. Citrullination of MBP, which functions in maintaining the stability of the myelin sheath (52,53), disrupts its tertiary structure, resulting in modified protein interactions, and possibly impaired cell adhesion properties (54,55). Citrullinated proteins like MBP are also more susceptible to proteolysis (56,57), which may generate antigenic determinants in the autoimmune response of demyelinating diseases (55,58–61). Citrullination also appears to inhibit cell proliferation, leading to cell cycle arrest and apoptosis (62, 63). We have hypothesized that citrullination contributes to glaucomatous neuropathy through changes in the dynamics of myelin components that likely disrupts myelination and the optic nerve head matrix protein framework.

Future PAD2 Studies

Current results implicate optic nerve PAD2 directed citrullination in glaucoma pathogenesis. Whether PAD2 expression and citrullination cause neurodegeneration in POAG or are consequential to damage in glaucomatous optic nerve remains to be determined. Of immediate interest is determining whether human retinal ganglion cells from POAG and normal donors exhibit differences in PAD2 levels and citrullination. Future studies of PAD2 expression and citrullination in additional glaucomatous and control animal models would be informative, including PAD2 over expression and knockdown analyses. PAD2 expression in POAG tissue appears to be regulated at the translational level, with calcium being a key factor (35). Additional studies are needed to compare the calcium concentration of glaucomatous and normal tissues and to probe the regulation of PAD2 in normal ocular tissues. The targeted degradation of PAD2 mRNA with shRNA in pressure treated astrocytes decreases PAD2 expression and citrullination. The therapeutic potential of such targeted approaches remains to be evaluated in appropriate animal models of glaucoma.