Dear Editor,

Prothymosin-α1 (ProTα), a highly acidic nuclear protein of the α-thymosin family, has multiple functions both within and outside of the cell.1 ProTα plays a cytoprotective role by inhibiting apoptosome formation,2 and it has cell proliferative activity as an extracellular signaling molecule.1 Recently, we found that ProTα is released upon necrotic stress and protects cells from neuronal death through activation of a putative Gi/o-coupled receptor coupled to phospholipase C and protein kinase CβII.3

Following stroke or traumatic damages, both necrotic and apoptotic neuronal death cause loss of functions including memory, sensory perception and motor skills.4, 5 Necrosis occurs during the early stages of stroke and of the affected area expands with time,6 whereas the apoptosis occurs with some delay, but may also have a potential role in restricting the spread of irretrievable damage.6, 7 There have been many attempts to find compounds that inhibit apoptosis, but the protective potencies of these compounds against ischemic damage in vivo remain limited.8, 9, 10, 11 This may be related to the possibility that rapid and expanding necrosis largely contributes to the total loss of brain neurons following ischemia. Thus, rapid treatment of stroke is currently emphasized.11, 12, 13 Compared with the machinery involved in apoptosis, necrosis is a more passive process, in which energy failure leads to a rupture of the plasma membrane with concomitant loss of intracellular proteins and ions.6 However, no compounds that inhibit necrosis are known. Here, we demonstrate that ProTα has potential therapeutic utility against the necrosis component of stroke.

In the focal ischemic model of middle cerebral artery occlusion (MCAO, 1 h) followed by reperfusion in the rat, a marked loss of triphenyltetrazolium chloride (TTC) staining, which detects the presence of mitochondrial enzymes, was observed specifically in the ipsilateral regions of the cerebral cortex, hippocampus and striatum at 24 h after reperfusion (Figures 1a–1d). Systemic administrations of recombinant rat ProTα (rrProTα, 100 μg/kg, i.p.) at 30 min and 3 h after reperfusion, largely reversed this brain damage and suppressed ischemia-induced motor dysfunction, evaluated by a so-called clinical score,14 and lethality (Figures 1f and 1g). It should be noted that ProTα protected the brain from cell death and motor dysfunction when administered as a single injection (at 30 min or 3 h after reperfusion) or in a pair of later injections (at 3 and 6 h after reperfusion). In a sublethal global ischemic model using both common carotid artery occlusions (BCCAO, 30 min), there were marked losses of neurons in the pyramidal cell layers and dentate gyri of surviving mice at 28 days after occlusion (Figure 1(h). A single systemic (i.p.) injection of recombinant mouse ProTα (rmProTα) at 100 μg/kg 24 h after occlusion completely prevented brain damage, learning and memory deficits in the step-through passive avoidance task, and lethality (Figures 1i–k). Significant prevention was also observed with rmProTα at a very low dose, 1 μg/kg (i.p.).

Figure 1
figure 1

ProTα in brain ischemia. (a) Typical photographs of TTC staining of ischemic brain. Recombinant rat ProTα (rrProTα, 100 μg/kg: i.p.) was given to rats at 30 min and 3 h after MCAO (1 h), and TTC staining was carried out 24 h later. (be) ProTα-induced prevention of brain damage (b: striatum, c: hippocampus, d: cortex) and dysfunction (e) due to focal MCAO ischemia reperfusion stress. Results represent data from 5 to 10 animals. *P<0.05 versus sham, #P<0.05 versus MCAO+vehicle. (f, g) ProTα-induced prevention of motor dysfunction (f) and lethality (g) due to MCAO stress (8–12 rats in each group). Motor dysfunction was evaluated by clinical score on each indicated day. rrProTα was given at 30 min and 3 h after MCAO. *P<0.05 versus sham, #P<0.05 versus ischemia alone. (h) Hematoxylin and eosin (HE) staining of hippocampal sections 28 days after sublethal BCCAO (30 min). Pictures of the whole hippocampus (left panel) and the CA1 region (right panel) are shown. (i, j) ProTα-induced prevention of brain damage (i) and dysfunction (j) by global BCCAO ischemia reperfusion stress. Number of cells in the CA1 region (0.2 × 0.2 mm; square region in Figure 1h, left panel). Results represent data from 5 to 10 animals. *P<0.05 versus sham, #P<0.05 versus BCCAO+vehicle. (k) Time course of the survival profile of mice given BCCAO with or without 24 h post-treatment with recombinant mouse ProTα (rmProTα, 100 μg/kg: i.p.). (l) ProTα-induced prevention of necrosis (propidium iodide, red) and apoptosis (activated caspase 3, green) 24 h after MCAO. (m–o) Quantitation of each cell death mode in each brain region (0.4 x 0.4 mm) was performed. *P<0.05 versus sham, #P<0.05 versus ischemia alone. (p, q) Reversal of ProTα-induced suppression of apoptosis by α-BDNF or α-EPO IgG. α-BDNF or α-EPO IgG (1 μg each) was given into the subarachnoid space through a parietal bone, 30 min before MCAO stress. Results represent the incidence of activated caspase 3 (green) and PI staining (red). *P<0.05. (r) Evidence for Myc-tagged ProTα transport through the blood–brain barrier after ischemic stress. Myc-tagged ProTα at 1 mg/kg (i.p.) was given to rats 30 min after reperfusion, and proteins (30 μg) from the cortex isolated 2.5 h later were used for Western blot analysis

By cytochemical analysis, both necrosis and apoptosis were observed in damaged brain regions 24 h after MCAO, when cell death was evaluated by in vivo propidium iodide and activated-caspase 3 staining. Systemic rrProTα injection (i.p.) markedly inhibited both necrotic and apoptotic cell death (Figures 1l–1o).

It should be noted that ProTα inhibited both necrosis and apoptosis following ischemia. This finding contrasts with our recent study, in which ProTα was shown to inhibit necrosis by restoring the reduced membrane translocation of glucose transporters under ischemic conditions in a primary culture of cortical neurons, but to cause apoptosis through upregulation of proapoptotic Bax and Bim.3 However, complete inhibition of cell death was observed when ischemic neurons were co-treated with ProTα and antiapoptotic neurotrophins, the expression levels of which are upregulated in in vivo ischemic models.15 In fact, the treatment with anti-BDNF or anti-EPO IgG (1 μg each) into the subarachnoid space through a parietal bone, 30 min before MCAO stress, reversed ProTα-inhibited apoptosis, but not necrosis (Figures 1p and q).

A single or paired injection of ProTα through a systemic route is sufficient to completely prevent damage in brain ischemic models. As shown in Figure 1r, a significant amount of Myc-tagged rrProTα, which had been administered i.p., was detected in the cortex 3 h after MCAO stress. The neuroprotective actions of ProTα administered through systemic routes are likely to be due to the transient disruption of the blood–brain barrier in the ischemic brain.16 It is worth to note that the neuroprotective actions of ProTα were observed when injected systemically 3 h after the ischemia, a time point within the therapeutic window for the treatment of stroke with tissue plasminogen activator. The fact that neurons are the major sites of action for ProTα is also expected to be advantageous in the treatment of stroke.

The paper by Shiau et al.17 described that added ProTα is incorporated into the cell and nuclei to ‘enhance’ the cytokine gene transcription and increase the infarct volume through inflammatory actions, whereas ProTα mutant lacking nuclear localizing sequence (NLS) ‘inhibits’ it. As shown in the Figure 1r, Myc-ProTα incorporated into the brain does not seem to be degraded and lose C-terminal NLS. Therefore, the finding by Shiau et al.17 is unlikely related to the ProTα-induced cell death inhibition in this study. Furthermore, in our previous study,3 there was no significant difference in the survival activity between ProTα and its mutant lacking C-terminal NLS.

Thus, ProTα is a unique cell death regulatory molecule, in that it converts irretrievable necrotic cell death into controllable apoptosis. Because this apoptosis can be inhibited by growth factors secreted upon ischemic stress, it is expected that ProTα may have an overall neuroprotective roles in the treatment of stroke.