Effect of calpain inhibitor XI on photoreceptor viability varies by treatment
We had previously established that, when included in the calpain assay system, CPI XI potently reduced the calpain activity that could be visualized in rd1 photoreceptors ex vivo (Paquet-Durand et al. 2006). We therefore tested the efficacy of this inhibitor to rescue rd1 photoreceptors in an in vitro organotypic retinal explant system. Two different treatment paradigms were utilized: chronic and acute, as described in Experimental Procedures (PN5 + 2 + 4 DIV and PN5 + 5 DIV + 16 h in vitro, respectively). The TUNEL assay for dying cells identified a significant, and concentration dependent, difference between the outcomes of the chronic and acute treatments (Fig. 1). As CPI XI concentration increased, the chronically-treated retinae showed more TUNEL-positive cells, particularly in the outer nuclear layer (ONL), but opposite to this, in acutely-treated retinae, their number decreased (Fig. 1a, e; b, f). Thus, at the highest CPI XI concentration (100 μM), chronic treatment resulted in a 195% increase in TUNEL-positivity in the ONL (8.3 ± 0.8% SEM, n = 4, p < 0.001) compared to vehicle treated (dimethylsulfoxide) rd1 retinae (4.3 ± 0.2%, n = 14), while after acute treatment the ONL of retinae exhibited 43% less TUNEL-positive cells (CPI XI treated: 2.5 ± 0.4%, n = 4; control: 4.4 ± 0.3%, n = 12, p < 0.01; Fig. 1i). The discrepancy between the effects of chronic and acute treatment was even more obvious when the percentage of TUNEL-positive cells was plotted against CPI XI concentration, and a linear regression fitted through the data points (Fig. 1j).
Figure 1. Acute CPI XI treatment preserves photoreceptors while chronic treatment induces cell death. The percentages of TUNEL-positive cells in the ONL of rd1 and wt retinae illustrate the drastic differences induced by 96 h chronic (a, e; c, g) and 16 h acute (b, f; d, h) inhibition. The quantification of this effect for different concentrations of CPI XI in rd1 and wt retinae (i) showed that as inhibitor concentration increased, chronic treatment caused more cell death, whereas photoreceptors were dose-dependently rescued after acute treatment. This strong discrepancy was also exemplified by linear regression analysis for rd1 photoreceptor cell death after acute and chronic treatment (j). Images shown are representative for the central area of retinal explant cultures. For each time-point retinae from at least four different wt or rd1 animals were analyzed. Error bars represent SEM, scale bar in (h) = 100 μm. Significance levels were: *p < 0.05, **p < 0.01, ***p < 0.001.
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Wild-type retinae responded to CPI XI treatment in similar ways (Fig. 1c, g; d, h). A comparatively low number of TUNEL-positive cells were seen in the vehicle treated ONL (1.6 ± 0.1%, n = 8), representing a low level of photoreceptor degeneration caused by the culture procedure. However, the levels of cell death were significantly increased by chronic CPI XI treatment (4.3 ± 0.4%, n = 10, p < 0.001), while acute treatment led to a numerical reduction of TUNEL-positive cells in the ONL (CPI XI treated: 0.9 ± 0.2%, n = 6; control: 1.5 ± 0.3%, n = 4, p = 0.08; Fig. 1i).
Calpastatin peptide protects rd1 photoreceptors in vitro
The results of the chronic CPI XI treatment prompted the need to look for a different, less toxic calpain inhibitor. When calpastatin peptide (CAST), at the concentration of 20 μM, replaced CPI XI under the identical chronic treatment, the percentage of TUNEL-positive cells in the rd1 ONL drastically diminished from 3.9% (± 0.4 SEM, n = 6) in untreated preparations to 1.8% (± 0.2, n = 12, p < 0.001) in CAST treated specimens. This was statistically equivalent to wt control (1.6 ± 0.1%, n = 8) and correlated with a statistically significant (p < 0.001) decrease of calpain activity-positive cells in the ONL (Fig. 2). Beyond the ONL, cell death labeling in the inner nuclear layer (INL) and ganglion cell layer of CAST-treated rd1 and wt retinae did not identify detrimental effects of CAST treatment on cultured tissue.
Figure 2. Calpastatin peptide reduces calpain activity and photoreceptor cell death in vitro: treatment of cultured retinal explants from PN7 to PN11 with 20 μM CAST resulted in a significant decrease in the percentage of TUNEL-positive cells (red bars in i) in the rd1 ONL (a, c), but not in wt (b, d). Similarly, the number of ONL cells showing high calpain activity (blue bars in i) was decreased by CAST (e, g). wt ONL was not affected by the treatment (f, h). The quantification (i) is based on cell counts obtained from at least three different wt or rd1 retinae. Error bars represent SEM. Images shown are representative for the central area of retinal explant cultures, bright staining sometimes visible in the upper part of an image relates to the culture membrane. Scale bar in (h) = 100 μm. Significance level was: ***p < 0.001.
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As judged by TUNEL analyses, CAST had a neuroprotective effect and CPI XI a photoreceptor toxic effect in the chronic treatment, in cultures ending at a time-point corresponding to PN11. However, at this time-point the rd1 degeneration had not yet led to an obvious decrease in the number of ONL cell rows, and by an experiment of this kind it was thus not possible to conclude that such treatments affect the long-term survival of photoreceptors. To study this, we prolonged the in vitro treatment to 14 DIV until a time-point corresponding to PN19 (PN5 + 2 + 12). Under these conditions, control wt retinae displayed 9.3 rows of photoreceptors in the ONL (± 0.3 SEM, n = 3; Fig. 3a) which was significantly decreased by CPI XI (40 μM) treatment to 6.8 (± 0.7, n = 3, p < 0.05; Fig. 3b). Vehicle treated degenerating rd1 retinae exhibited 3.3 (± 0.3, n = 9; Fig. 3c) rows of photoreceptors at PN19. This was further reduced by CPI XI treatment (2.8 ± 0.2, n = 6; Fig. 3d), although this difference did not attain statistical significance. In clear opposition to this, CAST (20 μM) treatment significantly increased the number of surviving rd1 photoreceptor rows by almost 70% (CAST: 5.5 ± 0.9, n = 3, p < 0.05; Fig. 3e, quantification in 3f).
Figure 3. Effects of long-term calpain inhibitor treatment on photoreceptor survival: retinal explants from wt and rd1 were cultured for 14 days in vitro (PN5 + 2 + 12) and exposed to either CPI XI or CAST. H/E staining revealed a significant decrease in the number of surviving photoreceptor rows when wt retina was treated with 40 μM CPI XI (a, b), suggesting photoreceptor toxicity (quantification of cell rows given in f). Degenerating rd1 retina (c) was not significantly affected by CPI XI (d) but showed a considerable and significant increase in photoreceptor survival when treated with CAST (e). In cultured wt retina, immunofluorescence showed typical distribution of rhodopsin in rod outer segments with ONL cells occasionally showing accumulation of rhodopsin in the cytoplasm (g). A magnification of the area surrounded by a white rectangle is shown in (g-i). CPI XI treated wt retina showed a marked increase in rhodopsin immunoreactivity in the ONL (h, h-i). In cultured rd1 retina, rod outer segments and rhodopsin immunofluorescence were strongly decreased, while there was unusual photoreceptor neurite outgrowth into the INL (i, i-i). The latter phenomenon was not observed in CPI XI treated rd1 retina (j, j-i). CAST treatment did not alter the neurite outgrowth phenotype but increased photoreceptor survival, without apparent preservation of rod outer segments (k, k-i). A quantification of the calpain inhibition effect on photoreceptor neurite outgrowth is given (l). Images shown are representative for the central area of retinal explant cultures. For each time-point at least three different wt or rd1 retinae were analysed. Error bars represent SEM, scale bars in k = 100 μm, k-i = 50 μm. Significance levels were: *p < 0.05, **p < 0.01.
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To obtain a further index on the status of the photoreceptors, the expression of rhodopsin was analyzed (Fig. 3g–k). Healthy, wt retinae in vivo show rhodopsin expression restricted to the outer segments of rod photoreceptors (Fig. 3g), while rd1 retinae, where outer segments do not develop properly, present with a partial mislocalization in the cytoplasm of photoreceptors, that is, staining is here readily seen in the ONL (Fig. 3i) (Bowes et al. 1988; Sancho-Pelluz et al. 2010). In degenerating rd1 retinae cultured until PN19 the size of rod outer segments and rhodopsin immunofluorescence was strongly reduced when compared to wt (Fig. 3g, i). In wt retinae, CPI XI appeared to increase rhodopsin immunoreactivity in the ONL (Fig. 3g, h). In rd1 explants after long-term CAST treatment, survival of photoreceptors was increased as judged by the number of ONL cell rows (Fig. 3f) with most ONL cells expressing rhodopsin (Fig. 3i, k) thus confirming rod photoreceptor identity. In the untreated rd1 control, however, many of the remaining ONL cells were rhodopsin-negative and hence most likely cone photoreceptors. Still, we cannot exclude the possibility that among the rhodopsin-negative cells, in the untreated rd1 situation, there might have been occasional rods which had lost rhodopsin expression.
The use of the rhodopsin staining also illustrated a remarkable photoreceptor neurite outgrowth into the inner nuclear layer of the untreated rd1 specimens (20.9 ± 2.4 neurites/mm) when compared to wt retinae (5.0 ± 0.6 neurites/mm), which resembled what has been reported previously for degenerating human rod photoreceptors (Li et al. 1995). However, such rod neurite sprouting was strongly reduced in CPI XI treated rd1 retinae (11.2 ± 3.0 neurites/mm) but not affected by CAST treatment (24.3 ± 2.9 neurites/mm), suggesting a potential dependency of this process on the activity of only certain types of calpains (Fig. 3i–l).
Taken together, CAST treatment in both short- and long-term retinal culture demonstrated a strong pro-survival effect, suggesting a major contribution of calpastatin targets to mutation induced rd1 photoreceptor death. At the same time, chronic application of CPI XI clearly had detrimental effects on photoreceptors, while the inner retina appeared to be less, or not at all, affected.
Inhibition of calpain reduces calpain activity and cell death in vivo
To explore the outcome of calpain inhibition in vivo in rd1 mice, CAST was injected into the vitreous body of the eye. In the first series of experiments, CAST was injected at PN10 and the percentage of dying photoreceptors was assessed using the TUNEL assay at different time-points post-injection. When compared to untreated specimens (Fig. 4a), at 4 h post-injection there was no significant effect, but at 8 h and 16 h (Fig. 4b) post-injection a strong and significant decrease in the number of TUNEL-positive cells was observed (Fig. 4c). At 48 h an obvious difference between treated and untreated specimens could no longer be noted, presumably because of drug clearance and/or degradation. As the damage to the early post-natal mouse eye caused by intravitreal injections is substantial, repeated injection traumas would result in severely increased retinal cell death. The considerable delay in the neuroprotective effects of CAST treatment in vivo could be because of the fact that at the time of treatment a certain number of cells were already compromised to an extent that did not allow rescue anymore.
Figure 4. CAST treatment reduces calpain activity and cell death in vivo: CAST (20 μM) was injected into the vitreous body at PN10. When compared to untreated rd1 retina, 16 h following CAST treatment the number of cells positive for cell death (TUNEL assay; a, b) and calpain activity (c, d) was strongly reduced. The merged image (e, f) illustrates the high degree of colocalization between TUNEL (red) and calpain activity (blue) positive cells. The protective effect of CAST treatment was maximal at 16 h post-injection (g). At this time-point, CAST decreased the number of cells positive for calpain (blue bars) and cell death (red bars) significantly (h). For each time-point at least three different rd1 animals were analyzed, error bars represent SEM, scale bar in (b) = 100 μm. Significance levels were: *p < 0.05, **p < 0.01.
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As the beneficial effect of the treatment was most pronounced at 16 h post-injection, in a second set of experiments the effects of CAST treatment on calpain activity and TUNEL assay were assessed using this treatment-analysis interval, which hence ends the test at an age roughly corresponding to PN11. When compared to untreated or sham-treated specimens, CAST injection in vivo led to a clear decrease in the number of cells positive for either calpain activity (rd1 CAST: 1.5 ± 0.3%, n = 3; rd1 sham: 3.6 ± 0.5%, n = 4, p < 0.01) or for TUNEL (rd1 CAST: 2.1 ± 0.2%, n = 3; rd1 sham: 3.2 ± 0.2%, n = 4, p < 0.01).
The reduction in the percentage of dying, TUNEL-positive cells in the rd1 ONL in vivo corresponds to the previous in vitro observations and thus confirms the neuroprotective effects of CAST treatment.