Increased cerebral protein ISGylation after focal ischemia is neuroprotective

Abstract

Addition of a small peptide called ISG15 is known as ISGylation, which is an ubiquitin (ub)-like posttranslational modification. We currently show that focal ischemia induced by transient middle cerebral artery occlusion (MCAO) in adult mice significantly induces cortical protein ISGylation between 6 and 24 hours reperfusion. With two-dimensional western blotting, 45 proteins were observed to be significantly increased in ISGylation (by 1.8- to 9.7-fold) after focal ischemia compared with sham control. Immunochemistry showed that ISGylated proteins are localized in neurons within the ipsilateral striatum and in astroglia within the peri-infarct cortex of ischemic mice. When subjected to transient MCAO, ISG15(-/-) mice showed increased mortality, exacerbated infarction, and worsened neurologic recovery than did wild-type controls. In addition, mice lacking UBE1L (ub-activating enzyme E1-like protein, the first enzyme of the ISGylation cycle) also showed bigger infarcts when subjected to transient MCAO. Regional cerebral blood flow or other physiologic parameters were not significantly different in both knockouts compared with wild-type controls. These studies indicate that increased protein ISGylation might be an endogenous neuroprotective adaptation to minimize poststroke brain damage.

Figure 1

Transient MCAO increased the ISGylation of proteins in the molecular weight range of 25 to 200 kDa (A). Protein levels of the E1 ligase of ISGylation cycle UBE1L significantly elevated after transient MCAO (B). Representative western blots were shown in the figure. Similar results were observed with n=6 animals per group. Histogram shows increased ISGylation and UBE1L after transient MCAO (C). 2D western blots of proteins extracts from the ipsilateral cortex of representative sham (left gel) and MCAO samples (right gel) was shown in panel D. In each case, 200 μg of total protein was used for electrophoresis. The gels were blotted onto PVDF membranes and probed with ISG15 antibodies. Molecular weight standards used for electrophoresis were myosin (220 kDa), phosphorylase A (94 kDa), catalase (60 kDa), actin (43 kDa), carbonic anhydrase (29 kDa), and lysozyme (14 kDa). Tropomyosin (33 kDa; pI 5.2) was used as an externally added reference. Of the 186 spots (ISGylated proteins reacted with ISG15 antibodies) analyzed by densitometric scanning, 51 were observed to be significantly different between the sham and MCAO groups (Supplementary Table 1). MCAO, middle cerebral artery occlusion; PVDF, polyvinylidene fluoride; 2D, two dimensional.

Figure 2

Colocalization of ISG15 protein expression with neuronal (NeuN) and astroglial (GFAP) markers in ischemic brain after 40 minutes MCAO, followed by 24 hours reperfusion. Double fluorescence labeling with rabbit polyclonal anti-ISG15 (top and bottom row, green), and mouse monoclonal anti-NeuN (top row red), or mouse monoclonal anti-GFAP (bottom row, red) was performed. Neuronal images were taken from the ipsilateral striatum and astrocytic images were taken from the peri-infarct cortex. Colocalization of ISG15 with NeuN (top row) and GFAP (bottom row) was shown by arrows. Scale bar for top and bottom rows is 31.1 and 47 μm, respectively. GFAP, glial fibrillary acidic protein; MCAO, middle cerebral artery occlusion. The color reproduction of this figure is available on the html full text version of the article.

Figure 3

Cresyl violet-stained serial brain sections (35 μm thick) from representative wild-type, ISG15^−/−, and UBE1L^−/− mice subjected to 40 minutes transient MCAO and 3 days reperfusion (top panel). The rCBF was not significantly different between the three genotypes during and after transient MCAO (middle panel). Both ISG15^−/− and UBE1L^−/− mice showed significantly higher infarct volume than did wild-type mice (bottom panel). ^*P<0.05 compared with wild-type (one-way ANOVA followed by the Tukey–Kramer multiple comparisons posttest). Values in middle and bottom panels are mean±s.d. (n=9 per group). ANOVA, analysis of variance; MCAO, middle cerebral artery occlusion; rCBF, regional cerebral blood flow.

Figure 4

(A) Immunostaining for ISG15 in the striatum and peri-infarct cortex of ischemic hemisphere of wild-type (top row), ISG15^−/− (middle row), and UBE1L^−/− (bottom row) mice after 40 minutes MCAO and 24 hours reperfusion. Western blot shows the ISG15 conjugates in the cortical lysates of wild-type, ISG15^−/−, and Ube1L^−/− after 40 minutes MCAO followed by 24 hours reperfusion. (B) The antibody is specific for ISGylated proteins except for one nonspecific band at ∼50 kDa in all samples. (C, D) Western blot analysis of the UBE1L protein in the cortical lysates of wild-type, UBE1L^−/−, and ISG15^−/− mice after 40 minutes MCAO and 24 hours reperfusion. MCAO, middle cerebral artery occlusion.