Extracellular α-crystallin protects astrocytes from cell death through activation of MAPK, PI3K/Akt signaling pathway and blockade of ROS release from mitochondria.

α-Crystallin with two isoforms, αA-crystallin (HSPB4) and αB-crystallin (HSPB5), is found in eye lens, spleen, lung, kidney, cornea, skin, but also in brain. Several studies revealed roles of αA/αB-crystallin in regulating cell viability and protection in the central nervous system. We previously demonstrated that α-crystallin serves as an intracellular protectant in astrocytes. Compared to well-studied intracellular functions of α-crystallin, there is limited proof for the role of α-crystallin as extracellular protectant. In order to clarify protective effects of extracellular αA/αB-crystallin, we exposed astrocytes to the toxic agents, staurosporine or C2-ceramide, or serum-starvation in the presence of αA/αB-crystallin. Extracellular αA/αB-crystallin protected astrocytes from staurosporine- and C2-ceramide-induced cell death. In addition, extracellular αB-crystallin/HSPB5 effectively promoted astrocytes viability through phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinases (JNK) signaling pathways under serum-deprivation. Furthermore, αB-crystallin/HSPB5 decreases the staurosporine-mediated cleavage of caspase 3 through PI3K/Akt signaling preventing apoptosis of astrocytes. Thus, the current study indicates that extracellular αA/αB-crystallin protects astrocytes exposed to various harmful stimuli. Furthermore, application of αB-crystallin/HSPB5 to isolated rat brain mitochondria inhibits ROS generation induced by complex III inhibition with Antimycin A.

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.

Latency antigen α-crystallin based vaccination imparts a robust protection against TB by modulating the dynamics of pulmonary cytokines.

BACKGROUND: Efficient control of tuberculosis (TB) requires development of strategies that can enhance efficacy of the existing vaccine Mycobacterium bovis Bacille Calmette Guerin (BCG). To date only a few studies have explored the potential of latency-associated antigens to augment the immunogenicity of BCG. METHODS/PRINCIPAL FINDINGS: We evaluated the protective efficacy of a heterologous prime boost approach based on recombinant BCG and DNA vaccines targeting α-crystallin, a prominent latency antigen. We show that "rBCG prime-DNA boost" strategy (R/D) confers a markedly superior protection along with reduced pathology in comparison to BCG vaccination in guinea pigs (565 fold and 45 fold reduced CFU in lungs and spleen, respectively, in comparison to BCG vaccination). In addition, R/D regimen also confers enhanced protection in mice. Our results in guinea pig model show a distinct association of enhanced protection with an increased level of interleukin (IL)12 and a simultaneous increase in immuno-regulatory cytokines such as transforming growth factor (TGF)ß and IL10 in lungs. The T cell effector functions, which could not be measured in guinea pigs due to technical limitations, were characterized in mice by multi-parameter flow cytometry. We show that R/D regimen elicits a heightened multi-functional CD4 Th1 cell response leading to enhanced protection. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based R/D regimen over BCG. Our observations from guinea pig studies indicate a crucial role of IL12, IL10 and TGFß in vaccine-induced protection. Further, characterization of T cell responses in mice demonstrates that protection against TB is predictable by the frequency of CD4 T cells simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and IL2. We anticipate that this study will not only contribute toward the development of a superior alternative to BCG, but will also stimulate designing of TB vaccines based on latency antigens.

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).

The neuroprotective efficacy of alpha-crystallin against acute inflammation in mice.

Acute inflammation activates macrophages or monocytes and subsequently releases several inflammatory cytokines and reactive oxygen and nitrogen species. These proinflammatory cytokines activate astrocytes and trigger neurodegenerative diseases. In this work, we chose to address the mechanistic aspects of alpha-crystallin's protective function in inflammation-triggered neurotoxicity in mice. Alpha-crystallin, a lens structural protein, comprising alpha-A and alpha-B subunits is an ubiquitous molecular chaperone, which have been shown to reduce reactive oxygen species (ROS) production and enhance cellular glutathione level in the acute inflammation-induced mice. Results show that the proinflammatory cytokines such as interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) were significantly high (P<0.05) in the plasma, liver, cortex and hippocampus of inflammation-induced mice when compared to control. Alpha-crystallin pretreatment prevents inflammation-induced cytokines and NO production. In addition, a significant (P<0.05) reduction of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) was also observed in the inflammation-induced mice. Nevertheless, their metabolites, such as 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) increased significantly (P<0.05) as compared to control. The results indicate that alpha-crystallin pretreatment controls the inflammation-induced DA, 5-HT and NE catabolism and suggest that alpha-crystallin has the potential to act as an anti-inflammatory agent in the neuroprotective processes.