Connexin 30 expression and frequency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.

We investigated age-associated changes in retinal astrocyte connexins (Cx) by assaying Cx numbers, plaque sizes, protein expression levels and heterogeneity of gap junctions utilizing six-marker immunohistochemistry (IHC). We compared Wistar rat retinal wholemounts in animals aged 3 (young adult), 9 (middle-aged) and 22 months (aged). We determined that retinal astrocytes have gap junctions composed of Cx26, -30, -43 and -45. Cx30 was consistently elevated at 22 months compared to younger ages both when associated with parenchymal astrocytes and vascular-associated astrocytes. Not only was the absolute number of Cx30 plaques significantly higher (P<0.05) but the size of the plaques was significantly larger at 22 months compared to younger ages (p<0.05). With age, Cx26 increased significantly initially, but returned to basal levels; whereas Cx43 expression remained low and stable with age. Evidence that astrocytes alter connexin compositions of gap junctions was demonstrated by the significant increase in the number of Cx26/Cx45 gap junctions with age. We also found gap junctions comprised of 1, 2, 3 or 4 Cx proteins suggesting that retinal astrocytes use various connexin protein combinations in their gap junctions during development and aging. These data provides new insight into the dynamic and extensive Cx network utilized by retinal astrocytes for communication within both the parenchyma and vasculature for the maintenance of normal retinal physiology with age. This characterisation of the changes in astrocytic gap junctional communication with age in the CNS is crucial to the understanding of physiological aging and age-related neurodegenerative diseases.

Tight binding of proteins to membranes from older human cells.

The lens is an ideal model system for the study of macromolecular aging and its consequences for cellular function, since there is no turnover of lens fibre cells. To examine biochemical processes that take place in the lens and that may also occur in other long-lived cells, membranes were isolated from defined regions of human lenses that are synthesised at different times during life, and assayed for the presence of tightly bound cytosolic proteins using quantitative iTRAQ proteomics technology. A majority of lens beta crystallins and all gamma crystallins became increasingly membrane bound with age, however, the chaperone proteins alpha A and alpha B crystallin, as well as the thermally-stable protein, ßB2 crystallin, did not. Other proteins such as brain-associated signal protein 1 and paralemmin 1 became less tightly bound in the older regions of the lens. It is evident that protein-membrane interactions change significantly with age. Selected proteins that were formerly cytosolic become increasingly tightly bound to cell membranes with age and are not removed even by treatment with 7 M urea. It is likely that such processes reflect polypeptide denaturation over time and the untoward binding of proteins to membranes may alter membrane properties and contribute to impairment of communication between older cells.

Age-dependent denaturation of enzymes in the human lens: a paradigm for organismic aging?

Little is known about the rate of denaturation of proteins within the human body. To monitor this decline, human eye lenses were dissected into discrete regions that were formed at different stages of life and assayed for activity of lactate dehydrogenase (LDH) and a particularly stable enzyme, glutathione reductase (GR). Activity was highest for both enzymes in the most recently synthesized outer part of the lens, decreased further into the lens, and, for LDH, was barely detectable in nuclear regions that consist of proteins that were synthesized in utero. For LDH, 95% of total lens activity was found in the outer half of the adult lens at all ages. Activity was unchanged in the outermost part of the lens as a function of age, suggesting that the ability of humans to synthesize the two enzymes is not impaired, even up to the tenth decade. After age of 40, LDH activity declined steadily in the interior of the lens at the rate of 8.3% per decade. GR activity diminished more slowly, and western blotting indicated that both denaturation of the enzyme and truncation were responsible. These data support the view that few, if any, metabolic pathways remain in the center of older lenses. Exposure of the enzymes to physiological pH and temperature over a period of decades is presumably sufficient to cause denaturation. The center of older human lenses is a unique environment in which the accumulation of untoward posttranslational modifications to proteins can be studied in the absence of significant enzymatic amelioration.

Protein aging: truncation of aquaporin 0 in human lens regions is a continuous age-dependent process.

The human lens is ideal for the study of macromolecular aging because cells in the centre, along with their constituent proteins, are present for our entire lives. We examined the major membrane protein, aquaporin 0 (AQP0), in regions of the lens formed at different times during our lifespan, to determine if similar changes could be detected and if they were progressive. Membrane fractions from three concentric lens regions were examined by SDS-PAGE coupled with densitometry, and Western blotting, to assess the time course of truncation. The overall extent of modification was also examined by MALDI mass spectrometry of the undigested proteins. In all regions, AQP0 became progressively more truncated, specifically by the loss of a 2kDa intracellular C-terminal peptide. The proteolysis increased steadily in all regions such that half of the AQP0 in the barrier region (that part of the lens formed immediately after birth) had been cleaved by age 40-50. MALDI mass spectrometry revealed that in all regions, AQP0 not only was shortened, it also became progressively more heterogeneous with age. Since the lens interior is devoid of active enzymes, it is very likely that the cleavage of AQP0 is chemically induced. We speculate that the loss of this C-terminal peptide 'spacer' may allow occlusion of AQP0 pores on the cytoplasmic face of the fibre cell membranes. Once a significant proportion of AQP0 has been cleaved, this occlusion may contribute to the formation of the lens permeability barrier that develops at middle age.

Reversible binding of kynurenine to lens proteins: potential protection by glutathione in young lenses.

PURPOSE: Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens. METHODS: The t-Boc-protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC. RESULTS: At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in beta-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys. CONCLUSIONS: These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.

Protein-bound and free UV filters in cataract lenses. The concentration of UV filters is much lower than in normal lenses.

In human cataract lenses the UV filters, 3-hydroxykynurenine glucoside (3OHKG) and kynurenine (Kyn) were found to be covalently bound to proteins and the levels in the nucleus were much higher than in the cortex. The levels of the bound UV filters in cataract nuclei were much lower than those in age-matched normal lenses. 3-Hydroxykynurenine could not be detected in cataract lenses. As with normal lenses, protein-bound 3OHKG in cataract lenses was found at the highest levels followed by Kyn. Free UV filter concentrations were also markedly reduced in cataract lenses. This feature may well contribute to the lower protein-bound levels; however, there was no clear relationship between free and bound UV filter contents when individual lenses were examined. We propose that since cysteine is a major site for UV filter binding, the well-documented oxidation of protein sulfhydryl groups during the progression of nuclear cataract may account, in part, for the pronounced decrease in bound UV filters in cataract lenses.

Protein-bound UV filters in normal human lenses: the concentration of bound UV filters equals that of free UV filters in the center of older lenses.

PURPOSE: To survey the levels of protein-bound UV filters in the cortices and nuclei of normal human lenses as a function of age and to relate this to the concentration of free UV filters. METHODS: Levels of each of the three kynurenine (Kyn) UV filters, 3-hydroxykynurenine glucoside (3OHKG), Kyn, and 3-hydroxykynurenine (3OHKyn), covalently attached to proteins, were determined by using a newly developed method of reductive capture, after base treatment of the intact lens proteins. RESULTS: The data show that, in the normal lens, each of the three UV filters became bound to proteins to a significant extent only after age 50 and, further, that the levels in the nucleus were much higher than in the cortex. These findings are consistent with the lens barrier that forms in middle age. 3OHKG was present at the highest levels followed by Kyn, with 3OHKyn being attached in the lowest amount. The ratio was 145:4:1 (3OHKG-Kyn-3OHKyn), with a total protein-bound UV filter concentration in the lens nucleus after age 50 of approximately 1300 picomoles/mg protein. This ratio is in agreement with 3OHKG being the most abundant free UV filter in the human lens and 3OHKyn being present in the lowest concentration with free Kyn present in intermediate amounts. CONCLUSIONS: The three Kyn UV filters are bound to the nuclear proteins of all normal lenses over the age of 50. Indeed in the center of older normal lenses, the concentration of UV filters bound to proteins is approximately equal to that of the free filters. Since bound UV filters promote oxidation of proteins after exposure to wavelengths of light that penetrate the cornea, lenses in middle-aged and older individuals may be more prone to photooxidation than those of young people.

3-Hydroxykynurenine oxidizes alpha-crystallin: potential role in cataractogenesis.

The alpha-, beta-, and gamma-crystallins are the major structural proteins of mammalian lenses. The human lens also contains tryptophan-derived UV filters, which are known to spontaneously deaminate at physiological pH and covalently attach to lens proteins. 3-Hydroxykynurenine (3OHKyn) is the third most abundant of the kynurenine UV filters in the lens, and previous studies have shown this compound to be unstable and to be oxidized under physiological conditions, producing H2O2. In this study, we show that methionine and tryptophan amino acid residues are oxidized when bovine alpha-crystallin is incubated with 3-hydroxykynurenine. We observed almost complete oxidation of methionines 1 and 138 in alphaA-crystallin and a similar extent of oxidation of methionines 1 and 68 in alphaB-crystallin after 48 h. Tryptophans 9 and 60 in alphaB-crystallin were oxidized to a lesser extent. AlphaA-crystallin was also found to have 3OHKyn bound to its single cysteine residue. Examination of normal aged human lenses revealed no evidence of oxidation of alpha-crystallin; however, oxidation was detected at methionine 1 in both alphaA- and alphaB-crystallin from human cataractous lenses. Age-related nuclear cataract is associated with coloration and insolubilization of lens proteins and extensive oxidation of cysteine and methionine residues. Our findings demonstrate that 3-hydroxykynurenine can readily catalyze the oxidation of methionine residues in both alphaB- and alphaA-crystallin, and it has been reported that alpha-crystallin modified in this way is a poorer chaperone. Thus, 3-hydroxykynurenine promotes the oxidation and modification of crystallins and may contribute to oxidative stress in the human lens.

Identification of 3-hydroxykynurenine bound to proteins in the human lens. A possible role in age-related nuclear cataract.

Age-related nuclear (ARN) cataract is a major cause of world blindness. With the onset of ARN cataract, the normally transparent and colorless lens becomes opaque and can take on colors ranging from orange, brown, and even black. The molecular basis for this remarkable transformation is unknown. ARN cataract is also characterized by extensive oxidation, insolubilization, and cross-linking of polypeptides, particularly in the nucleus of the lens. It has been postulated that 3-hydroxykynurenine (3OHKyn) may be involved in these changes. This endogenous tryptophan metabolite is readily oxidized and is involved in the tanning of moth cocoons and the formation of pigments in the eyes of butterflies. 3OHKyn is a component of our primate-specific UV-filter pathway, and the brownish hue of ARN cataract lenses is also unique to humans. Because numerous colored compounds can be produced by autoxidation of 3OHKyn, this process could provide an explanation for the variety of lens colors and other changes seen in ARN cataract. For such a theory to be tenable, it needs to be demonstrated that 3OHKyn is bound to proteins in the human lens. Here, we show that all normal lenses older than 50 have 3OHKyn covalently attached to the nuclear proteins, most likely via cysteine residues. If indeed 3OHKyn is implicated in ARN cataract, a reduction in the levels that are bound in cataract, compared to normal lenses, would be expected. In agreement with this hypothesis, no bound 3OHKyn could be detected in proteins isolated from ARN cataract lenses.