Protection against ischemic brain injury by protein therapeutics

Abstract

Preventing massive cell death is an important therapeutic strategy for various injuries and disorders. Protein therapeutics have the advantage of delivering proteins in a short period. We have engineered the antiapoptotic bcl-x gene to generate the super antiapoptotic factor, FNK, with a more powerful cytoprotective activity. In this study, we fused the protein transduction domain (PTD) of the HIVTat protein to FNK and used the construct in an animal model of ischemic brain injury. When added into culture media of human neuroblastoma cells and rat neocortical neurons, PTD-FNK rapidly transduced into cells and localized to mitochondria within 1 h. It protected the neuroblastomas and neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, respectively. The cytoprotective activity of PTD-FNK was found at concentrations as low as 0.3 pM. Additionally, PTD-FNK affected the cytosolic movement of calcium ions, which may relate to its neuroprotective action. Immunohistochemical analysis revealed that myc-tagged PTD-FNK (PTD-myc-FNK) injected i.p. into mice can have access into brain neurons. When injected i.p. into gerbils, PTD-FNK prevented delayed neuronal death in the hippocampus caused by transient global ischemia. These results suggest that PTD-FNK has a potential for clinical utility as a protein therapeutic strategy to prevent cell death in the brain.

Fig 1.

(A) Transduction of PTD-FNK into neuroblastoma SH-SY5Y cells. At the time indicated after the addition of 30 nM PTD-FNK, cells were washed once with PBS and fixed. Ten minutes before fixation, 100 nM MitoTracker red was added as the mitochondria indicator. PTD-FNK was immunodetected with anti-Bcl-x antibody (green) and colocalized with mitochondria (red). (Scale bar: 20 μm.) (B) Transduction of PTD-FNK into primary cultured neocortical neurons. Thirty minutes after adding PTD-FNK at the indicated concentrations, cells were washed once with PBS and fixed. Ten minutes before fixation, 100 nM MitoTracker red was added. Immunoreactivity of PTD-FNK (green) localized with mitochondria (red). (Scale bar: 20 μm.) (C) Quantification of the incorporated PTD-FNK protein in SH-SY5Y and primary neocortical neurons. The fluorescent intensity (arbitrary unit) of each cell in A (Left) and B (Right) were estimated by using the nih image program to obtain averages with SD (vertical bars). *, P < 0.003, compared with time 0 h (Left) and with no PTD-FNK (Right) by one-way ANOVA. (D) Decay of PTD-FNK incorporated into SH-SY5Y cells. Cells were incubated with 30 nM PTD-FNK for 1 h. After being washed once with DMEM, the cells were incubated in the medium with FBS for indicated periods, followed by fixation for immunostaining with anti-Bcl-x. Fluorescence of the cells was imaged with a confocal scanning microscope. The fluorescent images (four fields of view, each field containing five cells) were quantified with nih image. The intensity of cells without PTD-FNK was used as a background to be subtracted from those of cells with PTD-FNK. The average at 0 h is taken as 100%. Vertical bar, SD.

Fig 2.

(A) Dose-dependent cytoprotective effect of PTD-FNK on STS-induced apoptosis of neuroblastoma SH-SY5Y cells. One hour after PTD-FNK addition at the concentrations indicated, STS was added. The cells were incubated for another day. Surviving cells were counted by staining with PI and Hoechest 33342. The mean of three to four independent experiments is presented with SD (vertical bars). *, P < 0.01; **, P < 0.001, compared with no PTD-FNK by one-way ANOVA. (B) Comparison between PTD-FNK and PTD-Bcl-x_L in the cytoprotective activity against STS. One hour after the addition of 30 pM of each protein (PTD-FNK, PTD-Bcl-x_L) or buffer (Ctl), STS was added at the concentrations indicated. Cells were incubated for 1 more day and stained with PI (red) and Hoechest 33342. The mean of three to four independent experiments is presented with SD (vertical bars). *, P < 0.05; **, P < 0.0001, compared with buffer only by one-way ANOVA. (C–E) Representative pictures of apoptotic nuclei (indicated by arrows) with (D and E) or without (C) 800 nM of STS (×200). One hour before the addition of STS, 300 pM PTD-FNK was added (E). The cells were incubated for 1 day and stained with PI (red) and Hoechst 33342 (blue).

Fig 3.

(A) Dose-dependent neuroprotective effect of PTD-FNK on excitotoxic cell death in primary cultured neocortical neurons induced to glutamate. Two hours after addition of PTD-FNK at the concentrations indicated, neocortical neurons were exposed to 1 mM glutamate for 1 h. The cells were washed and incubated in medium for 1 day. The number of viable neurons was counted under a phase-contrast microscope in five fields of view (FOV) per well for four independent wells, and the average is presented with SD (vertical bars). *, P < 0.05; **, P < 0.001, compared with 0 pM by one-way ANOVA. (B) Comparison between PTD-FNK and PTD-Bcl-x_L in the neuroprotective activity against glutamate. Neocortical neurons were incubated with 30 pM of each protein (PTD-FNK, PTD-Bcl-x_L, and FNK) or buffer only (Ctl) for 2 h. Washed cells were exposed to glutamate as described in A. After 1 day, the number of viable neurons was counted under a phase-contrast microscope in five FOV per well for four independent wells. The average is presented with SD (vertical bars). *, P < 0.05, compared with no protein (Ctl) by one-way ANOVA. (C) Representative images of viable neurons observed in A by immunostaining with anti-MAP2 antibody (×100). (D) Delayed [Ca²⁺]_i deregulation in neurons exposed to glutamate. Representative tracings of changes in [Ca²⁺]_i are shown. Neurons were pretreated with or without PTD-FNK (3 nM) for 2 h in medium. Washed cells were placed in HBSS containing 2 mM Ca²⁺ and Fluo-3 AM. Twenty individual cells were assessed by confocal scanning fluorescent microscopy. Many neurons without PTD-FNK showed a secondary delayed increase in [Ca²⁺]_i indicated by * (Left), after the initial response induced by the addition of glutamate (arrows). In most neurons treated with PTD-FNK, the secondary increase did not occur (Right).

Fig 4.

(A and B) Transduction of myc-tagged PTD-FNK proteins into neurons in the brain. PTD-myc-FNK (50 mg/kg) (A) or vehicle (B) was injected i.p. into mice. After 10 h brains were perfused transcardially with 4% paraformaldehyde. Paraffin sections (4 μm) were incubated with a rabbit anti-Myc Tag polyclonal antibody. The antibody complex was detected with a DAKO Envision⁺ system. Cortices are shown. (Scale bars: A and B, 50 μm.) (C–H) PTD-FNK reduces cerebral ischemic damage. Gerbils injected with vehicle (n = 8) or PTD-FNK (5 mg/kg; n = 5) were subjected to ischemic insults for 5 min. After 7 days, animals were perfused transcardially with 4% formaldehyde and 5-μm thick cross sections containing hippocampal tissue were stained with hematoxylin–eosin. (C) Neuronal cell density in the hippocampal CA1 region of gerbils injected with vehicle (Ctl) or PTD-FNK. The number of surviving CA1 neurons is shown as intact neurons per 1-mm CA1 length (neuronal cell density; see Materials and Methods). The horizontal bar represents the average of neuronal cell density in gerbils injected with PTD-FNK. The average in gerbils without ischemic insult (untreated; n = 3) is 196 cells per mm. The histopathological damage (percentage dead neurons) was statistically assessed by the Mann–Whitney U test. (D–K) The hippocampus stained with hematoxylin–eosin. The hippocampus of a gerbil injected with vehicle (D and E) or PTD-FNK (F–I). The hippocampus of a gerbil without ischemic insult is also shown (J and K). The boxes in D, F, H, and J have been enlarged in E, G, I, and K, respectively. (Scale bars: D, F, H, and J, 250 μm; E, G, I, and K, 50 μm.) The number of surviving CA1 neurons in F and H was 35 and 174 cells per mm, respectively, and is shown in C.