The Small Heat Shock Protein α-Crystallin B Shows Neuroprotective Properties in a Glaucoma Animal Model

Abstract

Glaucoma is a neurodegenerative disease that leads to irreversible retinal ganglion cell (RGC) loss and is one of the main causes of blindness worldwide. The pathogenesis of glaucoma remains unclear, and novel approaches for neuroprotective treatments are urgently needed. Previous studies have revealed significant down-regulation of α-crystallin B as an initial reaction to elevated intraocular pressure (IOP), followed by a clear but delayed up-regulation, suggesting that this small heat-shock protein plays a pathophysiological role in the disease. This study analyzed the neuroprotective effect of α-crystallin B in an experimental animal model of glaucoma. Significant IOP elevation induced by episcleral vein cauterization resulted in a considerable impairment of the RGCs and the retinal nerve fiber layer. An intravitreal injection of α-crystallin B at the time of the IOP increase was able to rescue the RGCs, as measured in a functional photopic electroretinogram, retinal nerve fiber layer thickness, and RGC counts. Mass-spectrometry-based proteomics and antibody-microarray measurements indicated that a α-crystallin injection distinctly up-regulated all of the subclasses (α, β, and γ) of the crystallin protein family. The creation of an interactive protein network revealed clear correlations between individual proteins, which showed a regulatory shift resulting from the crystallin injection. The neuroprotective properties of α-crystallin B further demonstrate the potential importance of crystallin proteins in developing therapeutic options for glaucoma.

Keywords: experimental glaucoma; neuroprotection; proteomics; α-crystallin B.

Figure 1

IOP elevation as a result of the episcleral vein cauterization. The intraocular pressure (IOP) of all cauterized eyes rose significantly (*** p < 0.001, n = 11, mean ± SD, parametric t-test) two weeks after the surgical intervention. With rise of the IOP, the intravitreal (IV) injection of α-crystallin B and PBS was performed. No noticeable influence on the IOP was observed due to the injection. The untreated contralateral eyes remained unaffected in terms of IOP changes.

Figure 2

Quantification of RGC density in retinal flat-mounts. Elevation of the IOP resulted in an average loss of ∆ = 12% retinal ganglion cell (RGCs) in the α-crystallin B injected animals compared to the untreated contralateral eyes, while the RGC loss in the PBS-injected animals was about 25% (** p < 0.01, ns—not significant, n = 11, mean ± SD, one-way ANOVA).

Figure 3

Survey of the retinal nerve fiber layer thickness using optical coherence tomography. Untreated contralateral eyes showed a 6% decrease of the retinal nerve fiber layer thickness (RNFLT) during the time of the study. IOP elevation and α-crystallin B injection effectuated in a loss of ∆ = 8%, while PBS-injected eyes showed a decrease of ∆ = 22% with respect to retinal nerve fiber layer (RNFL) thickness (** p < 0.01, ns—not significant, n = 5, mean ± SD, one-way ANOVA).

Figure 4

Pattern of the ganzfeld photopic electroretinogram analysis. ERG results showed considerable aberrances in the B-wave and photopic negative response (PhNR) between contralateral control eyes and experimental glaucoma eyes. However, the deviance to the respective control eyes seems much higher in PBS-injected eyes than in α-crystallin B injected eyes (n = 11, mean ± SD). The millivolt decrease is defined in percentaged change (∆ = % change).

Figure 5

MS fold-change analysis of crystallin protein family members. Injection of α-crystallin B induced moderate to distinct up-regulation of α-, β-, and γ-crystallins in the retina when compared to PBS-injected retinal protein levels (** p < 0.01, * p < 0.05, ^# p < 0.1, unpaired parametric t-test, n = 4 per exp. group, mean ± SEM).

Figure 6

Ingenuity Pathway Analysis interactive protein network of highly up- or down-regulated retinal proteins. Up-regulated proteins (red) are located in the extracellular space, plasma membrane, cytoplasm, as well as in the nucleus. Down-regulated proteins in this network are exclusively located in the nucleus (green). The mitogen-activated protein kinase 1, protein kinase C (yellow) and caspase proteins show considerable interaction with a variety of displayed proteins with altered regulation levels. Legend: A = activation; L = proteolysis; P = phosphorylation/dephosphorylation; PP = protein–protein binding; I = inhibition; E = expression; PD = protein–DNA binding; TR = translocation; MB = group/complex membership; solid arrow = direct interaction; dashed arrow = indirect interaction; (count) = number of scientific references on the respective relation; if not indicated differently in the figure, the arrow head indicates “acts on”.

Figure 7

Antibody microarray detection of specifically altered retinal proteins. The protein regulations, detected in the MS measurements, could be validated with antibody microarray for lamin A/C, β-crystallin B2 and γ-crystallin C, using GAPDH for loading control purposes. Lamin A/C levels in the PBS group were significantly higher than in the crystallin-injected (cryab) group, while the levels of β-crystallin B2 and γ-crystallin C were found higher in the cryab group (* p < 0.05, ** p < 0.01, *** p < 0.001, ns—not significant, n = 9 per exp. group, mean ± SD, one-way ANOVA).