Effect of Sorbitol on Alpha-Crystallin Structure and Function.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

Latency-associated protein Acr1 impairs dendritic cell maturation and functionality: a possible mechanism of immune evasion by Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M. tuberculosis) in latently infected individuals survives and thwarts the attempts of eradication by the immune system. During latency, Acr1 is predominantly expressed by the bacterium. However, whether M. tuberculosis exploits its Acr1 in impairing the host immunity remains widely unexplored. Hence, currently we have investigated the role of Acr1 in influencing the differentiation and function of dendritic cells (DCs), which play a cardinal role in innate and adaptive immunity. Therefore, for the first time, we have revealed a novel mechanism of mycobacterial Acr1 in inhibiting the maturation and differentiation of DCs by inducing tolerogenic phenotype by modulating the expression of PD-L1; Tim-3; indoleamine 2, 3-dioxygenase (IDO); and interleukin 10. Furthermore, Acr1 interferes in the differentiation of DCs by targeting STAT-6 and STAT-3 pathways. Continuous activation of STAT-3 inhibited the translocation of NF-κB in Acr1-treated DCs. Furthermore, Acr1 also augmented the induction of regulatory T cells. These DCs displayed decline in their antigen uptake capacity and reduced ability to help T cells. Interestingly, M. tuberculosis exhibited better survival in Acr1-treated DCs. Thus, this study provides a crucial insight into a strategy adopted by M. tuberculosis to survive in the host by impairing the function of DCs.

Thermal denaturation and aggregation of apoform of glycogen phosphorylase b. Effect of crowding agents and chaperones.

The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70).

Alpha-crystallin promotes rat retinal neurite growth on myelin substrates in vitro.

BACKGROUND: Myelin-associated molecules are major impediments to axon regeneration after optic nerve injury. Intravitreal injection of α-crystallin can protect axons from optic nerve degeneration after crushing in rats. OBJECTIVES: Our purpose was to investigate whether α-crystallin could counteract the inhibitory effect of myelin and promote neurite growth. METHODS: Newborn rat retinal neurons were cultured on myelin-coated dishes with DMEM containing α-crystallin (10(-4) g/l) or bovine serum albumin. The density of neurons with neurites and the length of the longest neurite of the cells were analyzed on days 1, 3 and 5. RESULTS: Cultures containing α-crystallin had significantly higher neurite-containing cell densities, and the neurites were significantly longer compared with cultures containing bovine serum albumin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors and stimulate neurite growth.

Aggregation of ß-crystallin through covalent binding to 1,2-naphthoquinone is rescued by α-crystallin chaperone.

Cataract induced by exposure to naphthalene is thought to mainly involve its metabolic activation, forming 1,2-naphthoquinone (1,2-NQ), which can modify proteins through chemical modifications. In the present study, we examined the effect of 1,2-NQ on aggregation of crystallins (cry) associated with cataract. Incubation of bovine ß-cry with 1,2-NQ caused covalent modification of ß-cry at Cys117 and Lys125 accompanied by reduction in its thiol content, resulting in a concentration- and temperature-dependent aggregation of ß-cry, whereas only little aggregation of α-cry induced by 1,2-NQ was seen. Interestingly, addition of α-cry to the reaction mixture of ß-cry and 1,2-NQ markedly blocked ß-cry aggregation induced by 1,2-NQ in a concentration-dependent manner. These results suggest that ß-cry predominantly undergoes chemical modification by 1,2-NQ, causing its aggregation, which is suppressed by the chaperone-like protein, α-cry. This ß-cry aggregation may be, at least in part, involved in the induction of cataract caused by 1,2-NQ.

Study of kinetics of thermal aggregation of mitochondrial aspartate aminotransferase by dynamic light scattering: protective effect of alpha-crystallin.

Thermal aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been studied at various temperatures and various protein concentrations by dynamic light scattering. The character of the dependence of protein aggregate size on time indicates that aggregation of mAAT proceeds in the regime of diffusion-limited cluster-cluster aggregation. Suppression of mAAT aggregation by alpha-crystallin is due to transition of the aggregation process into the regime of reaction-limited cluster-cluster aggregation. Realization of this regime of aggregation means that the sticking probability for the colliding particles is less than unity.

alpha-Crystallin downregulates the expression of TNF-alpha and iNOS by activated rat retinal microglia in vitro and in vivo.

PURPOSE: Inhibition of microglial activation has become an important strategy to attenuate neurotoxic damage to the central nervous system. We evaluated the effects of alpha-crystallin on the production of cytokines in lipopolysaccharides (LPS) and optic nerve injury-activated retinal microglia. METHODS: Microglia were collected from retinas of newborn rats, cultured and treated with LPS in vitro. Microglia were also activated by an optic nerve crush in vivo. Pretreatments with and without alpha-crystallin were performed in cultured cells, and by intravitreal injection in adult rats. Expression of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO) and inducible NOS synthase (iNOS) were measured by RT-PCR, ELISA, Western blot and the nitrate reductase method. RESULTS: Activated microglia significantly upregulated TNF-alpha and iNOS mRNA expression and protein production in vitro. An optic nerve crush also increased expression of retinal iNOS and TNF-alpha protein. Treatment with alpha-crystallin in vitro and in vivo downregulated their expression. CONCLUSION: The protective effect of alpha-crystallin may be due to its effect on microglia via a downregulation in the expression and release of 2 key immune regulatory and inflammatory molecules: TNF-alpha and iNOS.

Alpha-crystallin protected axons from optic nerve degeneration after crushing in rats.

In mature mammals, optic nerve injury results in apoptosis of retinal ganglion cells. The literature confirms that lens injury enhances retinal ganglion cells survival, but the mechanism is not very clear. Using silver staining method and computer image analysis techniques, the effect of alpha-crystallin, a major component of the lens in the survival of retinal ganglion cell axons, was investigated in vivo after intravitreal injections. The results showed that enhanced survival of axotomized axons was observed beyond the crush site after a single intravitreal administration of alpha-crystallin at the time of axotomy. Axonal density of the retinal ganglion cell was significantly greater than in the untreated controls until 2 weeks after injection. This effect declined by 4 weeks after injection but survival of axons remained greater than controls. These findings indicate that alpha-crystallin plays a key role in protecting axons after optic nerve injury.

Chaperone-mediated inhibition of tubulin self-assembly.

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self-assembly efficiently. Chaperones tested include alpha-crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and alpha (s)-casein from milk. All of them inhibit polymerization in a dose-dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self-assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and alpha (s)-casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 xx 10(4) and 9.8 xx 10(5) M(-1) for HSP16.3 and alpha (s)-casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones-tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin-chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein-protein interaction site involved in the nucleation phase of self-assembly.

Revival of glutathione reductase in human cataractous and clear lens extracts by thioredoxin and thioredoxin reductase, in conjunction with alpha-crystallin or thioltransferase.

Glutathione reductase (GR) plays a key role in maintaining thiol groups in the lens, and its activity decreases with aging and cataract formation. Mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), or the Trx/TrxR system, participates in the repair of oxidatively damaged lens proteins and enzymes. Alpha-crystallin, a molecular chaperone, prevents the aggregation of partially denatured proteins under various stress conditions. Thioltransferase (TTase, or glutaredoxin) can maintain the homeostasis of lens protein thiols thus protecting against oxidative stress. We investigated whether the Trx/TrxR system can revive GR activity in both the cortex and nucleus of human cataract and clear aged lenses and whether alpha-crystallin and TTase can help this effect. The GR activity in the cortex and nucleus of the cataractous lenses was significantly lower than that of the aged clear lenses. The highest activity in the cortex was observed in the clear aged lenses. The combination of Trx and TrxR revived the activity of GR from both the cortex and nucleus of aged clear lenses. However, in cataract lenses (grade II and grade IV), there was a statistically significant recovery of GR activity in the cortex, but not in the nucleus. No recovery was observed when Trx or TrxR were used separately. Alpha-crystallin successfully revived GR activity in the cortex of cataract grade II lenses, but not in the nucleus. The combination of alpha-crystallin and Trx/TrxR gave a further increase of activity. TTase alone revived some of the GR activity but together with the Trx/TrxR system gave no statistically significant enhancement of GR activity. These results indicate that both disulfide bond formation and protein unfolding are responsible for GR inactivation.

The protective effect of alpha-crystallin against acute inflammation in mice.

Acute inflammation can activate macrophages or monocytes and subsequently release several inflammatory cytokines and reactive oxygen species (ROS). Oxidative stress triggered by the production of ROS plays deleterious role leading to multiple organ failure. This study was designed to investigate the prophylactic effect of alpha-crystallin, a major chaperone lens protein comprising of alpha-A and alpha-B subunits in inflammation-induced mice. Mice were divided into three groups (n=6 in each): control, inflammation and alpha-crystallin-treated. Results show that ROS was significantly higher in the lymphocytes, hepatocytes and astrocytes (P<0.05) of inflammation-induced mice when compared to control, but no significant changes were observed in the alpha-crystallin-treated group. Increased level of lipid peroxidation (LPO) and decreased activities of antioxidant such as superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione were observed in the inflammation-induced mice when compared to control, whereas the activities of these were found to be normal followed by alpha-crystallin treatment. We also observed a reduction in reduced glutathione levels in hepatocytes of inflammation-induced mice, which were normalized on alpha-crystallin treatment. The in vitro study has shown that alpha-crystallin treatment not only suppresses the increase in LPO levels but also inhibits the lipid breakdown resulting from autooxidation in mouse cerebral cortex homogenate, and strongly suggests that alpha-crystallin therapy may serve as a potent pharmacological agent in systemic inflammation.

Thioredoxin, thioredoxin reductase, and alpha-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts.

PURPOSE: To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses. METHODS: The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-crystallin was investigated. RESULTS: GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, alpha(L)-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus. CONCLUSIONS: This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and alpha-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by unfolding, and can be recovered by reducing agents and a molecular chaperone.

Small heat shock proteins inhibit amyloid-beta protein aggregation and cerebrovascular amyloid-beta protein toxicity.

Small heat shock proteins Hsp20 and HspB2/B3 co-localize with Abeta deposition in senile plaques and cerebral amyloid angiopathy in Alzheimer's disease brains, respectively. It was the aim of our study to investigate if these and other sHsps bind to wild-type Abeta1-42 or the more toxic Abeta1-40 carrying the 'Dutch' mutation (22Glu-->Gln) (D-Abeta1-40), affect Abeta aggregation and thereby influence Abeta cytotoxicity. Binding affinity between sHsps and Abeta was investigated by surface plasmon resonance. Abeta aggregation was studied by using circular dichroism spectroscopy and electron microscopy. Furthermore, we used cultured cerebrovascular cells to investigate the effects of sHsps on Abeta-mediated cytotoxicity. Hsp20, Hsp27 and alphaB-crystallin, but not HspB2/B3, bound to Abeta (both D-Abeta1-40 and Abeta1-42) and reduced or completely inhibited aggregation of D-Abeta1-40 into mature fibrils but did not affect Abeta1-42 aggregation. Furthermore, these sHsps were effective inhibitors of the cerebrovascular toxicity of Abeta (both D-Abeta1-40 and Abeta1-42) in vitro. Binding affinity of the sHsps to D-Abeta1-40 correlated to the degree of inhibition of Abeta-mediated cytotoxicity and the potential to reduce Abeta beta-sheet and fibril formation. With Abeta1-42, a similar correlation between binding affinity and cytotoxicity was observed, but not with its aggregation state. In conclusion, sHsps may regulate Abeta aggregation and serve as antagonists of the biological action of Abeta, but the extent of their interaction depends on the type of sHsp and Abeta peptide.

Targeting the molecular mechanisms of ischemic damage: Protective effects of alpha-crystallin-B.

AIMS: Molecular chaperones constitute protectors of intracellular protein integrity and seem to confer short-term defence against various cell insults. Myocardial damage is associated to a loss of protective chaperones. Ischemic post-conditioning (IPost-Co) is a procedure that seems to protect against reperfusion injury. However, little is known on alpha-crystallin-B-chain (cryab/HspB5) evolution in IPost-Co. Here we have investigated cryab in myocardial ischemia and IPost-Co. METHODS AND RESULTS: Pigs underwent closed-chest 1.5h mid-left anterior descending (LAD) balloon occlusion and were either sacrificed without reperfusion (I;N=10), subjected to 2.5h of reperfusion and sacrificed (I/R; N=5); or subjected to IPost-Co before reperfusion and sacrificed 2.5h afterwards (IPost-Co; N=5). A sham-operated group was included (N=6). Proteomic analysis (2-D-electrophoresis/MALDI-TOF/TOF) revealed cryab as a single spot (20kDa; pI7.6). Myocardial cryab-20-protein and cryab-gene expression levels were decreased after ischemia and I/R(P<0.05). After IPost-Co, cryab-20-protein and cryab-gene expression levels were similar to those found in the heart of sham-operated animals (P<0.05). There was a direct correlation between LVEF-improvement after IPost-Co and myocardial cryab-20-protein levels. In a mice proof-of-principle study, cryab-20-peptide was synthesized and administered 1h before LAD-ligation and ECG-proven MI. A 59% reduction in infarct size was achieved in cryab-20-treated animals (P<0.05). CONCLUSIONS: Ischemia and reperfusion induce a decrease in myocardial cryab-20-protein levels together with a clinical impairment of cardiac function. IPost-Co induces a clinical improvement of cardiac function and a preservation of cryab-20 levels. Intervention studies on a mice-MI model showed that cryab-20-peptide administration reduces infarct size. All together our results show a significant cardioprotective effect of cryab.

Casein proteins as molecular chaperones.

Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.

Glutathione reductase from human cataract lenses can be revived by reducing agents and by a molecular chaperone, alpha-crystallin.

PURPOSE: The aim of this study was to investigate how glutathione reductase (GR) loses its activity during cataract formation and whether it is possible to revive it back to the normal levels. METHOD: In this study, endogenous as well as synthetic reducing systems (GSH, TTase, DTT, captopril) and alpha-crystallin at different concentrations were incubated with the soluble fraction of human cataract lens protein. The activity of glutathione reductase with or without the reducing agents and alpha-crystallin was tested, and the difference in activity gained was calculated. RESULTS: Five agents (GSH, DTT, TTase, captopril, alpha-low crystallin) were able to revive the activity of GR from human cataract lenses to different extents. CONCLUSION: This study shows that human lens GR activity was revived by different reducing agents as well as by a molecular chaperone (alpha-crystallin).

Quantification of anti-aggregation activity of UV-irradiated α-crystallin.

Ultraviolet radiation is a risk factor for cataractogenesis. It is believed that enhanced rates of lens opacification and cataract formation are the results of gradual loss of chaperone-like efficiency of α-crystallin upon exposure to UV light. To characterize chaperone-like activity of α-crystallin damaged by UV irradiation, a test system based on dithiothreitol-induced aggregation of holo-α-lactalbumin from bovine milk was used. The adsorption capacity of α-crystallin (AC0) with respect to the target protein (α-lactalbumin) was used as a measure of anti-aggregation activity of α-crystallin. The data on SDS-PAGE testify that UV irradiation of α-crystallin results in covalent cross-linking of subunits in α-crystallin oligomers. The dependence of AC0 value on the irradiation dose was compared with the UV-induced diminution of the portion of native α-crystallin estimated from the data on differential scanning calorimetry. On the basis of such comparison a conclusion has been made that the loss in chaperone-like activity is mainly due to UV-induced denaturation of α-crystallin subunits. Cross-linking of remaining native subunits leads to an additional decrease in anti-aggregation activity.

Study of the interaction between triplet riboflavin and the alpha-, betaH- and betaL-crystallins of the eye lens.

Time-resolved photolysis studies of riboflavin (RF) were carried out in the presence and absence of alpha-, betaH- and betaL-crystallins of bovine eye lens. The transient absorption spectra, recorded 5 micros after the laser pulse, reveal the presence of the absorption band (625-675 nm) of the RF neutral triplet state (tau = 42 micros) accompanied by the appearance of a long-lived absorption (tau = 320 micros) in the 500-600 nm region due to the formation of the semireduced RF radical. The RF excited state is quenched by the crystallin proteins through a mechanism that involves electron transfer from the proteins to the flavin, as shown by the decrease of the triplet RF band with the concomitant increase of the band of its semireduced form. Tryptophan loss on RF-sensitized photooxidation of the crystallins when irradiated with monochromatic visible light (450 nm) in a 5% oxygen atmosphere was studied. A direct correlation was found between the triplet RF quenching rate constants by the different crystallin fractions and the decomposition rate constants for the exposed and partially buried tryptophans in the proteins. The RF-sensitized photooxidation of the crystallins is accompanied by the decrease of the low molecular weight constituents giving rise to its multimeric forms. A direct correlation was observed between the initial rate of decrease of the low molecular weight bands corresponding to the irradiated alpha-, betaH- and betaL-crystallins and the quenching constant values of triplet RF by the different crystallins. The correlations found in this study confirm the importance of the Type-I photosensitizing mechanism of the crystallins, when RF acts as a sensitizer at low oxygen concentration, as can occur in the eye lens.

Lens alpha-crystallin and hypericin: a photophysical mechanism explains observed lens.

Determining whether alpha-crystallin (the major lens protein) affects the photophysics of hypericin, a photosensitizing agent found in various plants, such as St. John's Wort, is important. Hypericin shows promise in cancer and human immunodeficiency virus therapy but may harm individuals taking St. John's Wort extracts (for mild to moderate depression). Hypericin causes hypericism, which is characterized by cellular damage in light-exposed areas. Ocular tissues are at risk for photosensitized damage; thus, we investigated the effects on hypericin photophysics by alpha-crystallin. We measured the transient absorption spectra and the 1270 nm luminescence of singlet (1Deltag) oxygen produced from hypericin in the presence of alpha-crystallin. alpha-Crystallin complexes hypericin, extending the lifetime of its triplet excited state; the Stern-Volmer slope is negative, but not linear, after a saturation curve. Damage to the lens protein by hypericin is known to occur via singlet oxygen, which oxidizes methionine, tryptophan and histidine residues. Binding to alpha-crystallin does not inhibit singlet oxygen formation by hypericin. alpha-Crystallin reacts with singlet oxygen with a rate constant of 1.3 x 10(8) M(-1) s(-1). Thus, we anticipate that hypericin will be an effective photosensitizer in the lens.

α-Crystallin protects RGC survival and inhibits microglial activation after optic nerve crush.

AIMS: Activation of retinal microglial cells (RMCs) is known to contribute to retinal ganglion cell (RGC) death after optic nerve injury. The purpose of this study was to investigate the effects of intravenous injection of α-crystallin on RGC survival and RMC activation in a rat model of optic nerve crush. MAIN METHODS: RGCs were retrogradely labeled with fluorogold. Rats were intravenously injected with normal saline or α-crystallin (0.05g/kg, 0.5g/kg, and 5 g/kg) at 2, 4, 6, 8, 10, and 12 days after the optic nerve crush. Activated RMCs were characterized using immunofluorescence labeling with CD11b, and TNF-α and iNOS expression was detected using immunoblot analyses. We analyzed the morphology and numbers of RGC and RMC 2 and 4 weeks after injury using fluorescence and confocal microscopy. KEY FINDINGS: The number of RGCs decreased after optic nerve injury, accompanied by significantly increased numbers of activated RMCs. Intravenous injection of α-crystallin decreased the number of RMCs, and enhanced the number of RGCs compared to saline injection. α-Crystallin administration inhibited TNF-α and iNOS protein expression induced by optic nerve injury. SIGNIFICANCE: Our results suggest that α-crystallin promotes RGC survival and inhibits RMC activation. Intravenous injection of α-crystallin could be a possible strategy for the treatment of optic nerve injury.