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Abstract

Calcium toxicity remains the central focus of ischemic brain injury. Calcium channel antagonists have been reported to be neuroprotective in ischemic animal models but have failed in clinical trials. Rather than block the calcium channels, calbindin proteins can buffer excessive intracellular Ca²⁺ as a result, maintain the calcium homeostasis. In the present study, we investigated the effect of calbindin D 28k (CaBD) in ischemic brain using the novel technique protein transduction domain (PTD)-mediated protein transduction. We generated PTD-CaBD in Escherichia coli, tested its biologic activity in N-methyl-d-aspartate (NMDA)- and oxygen—glucose deprivation (OGD)-induced hippocampal injury models, and examined the protection of the fusion protein using a rat brain focal ischemia model. Infarct volume was determined using 2,3,5-triphenyl-tetrazolium chloride staining; neuronal injury was examined using terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining and cleaved caspase-3 assay. The results showed that the PTD-CaBD was efficiently delivered into Cos7 cells, hippocampal slice cells, and brain tissue. Pretreatment with PTD-CaBD decreased intracellular free calcium concentration and reduced cell death in NMDA- or OGD-exposed hippocampal slices (P < 0.05). Introperitoneal administration of PTD-CaBD before transient middle cerebral artery occlusion decreased brain infarct volume (280 ± 47 versus 166 ± 70 mm³ P < 0.05), and improved neurologic outcomes compared with the control. Further studies showed that, compared with the control animals, PTD-CaBD decreased TUNEL (58% ± 7% versus 29% ± 3%, P < 0.05)- and cleaved caspase-3 (62 ± 4/field versus 31 ± 6/field, P < 0.05)-positive cells in the ischemic boundary zone. These results indicate that systemic administration of PTD-CaBD could attenuate ischemic brain injury, suggesting that PTD-mediated protein transduction might provide a promising and effective approach for the therapies of brain diseases, including cerebral ischemia.

Keywords

apoptosis blood—brain barrier brain calbindin D 28k focal ischemia protein transduction

Introduction

Intracellular calcium (Ca²⁺) overloading plays a critical role in ischemic brain injury. Normally, calcium is present at a ratio of 10,000:1 between the extracellular fluid and the intracellular cytosol (Berridge et al, 2003); however, when ischemia occurs, excessive Ca²⁺ accumulates in the cytosol and mitochondria because of the rapid exhaustion of ATP supply in the brain. The superfluous Ca²⁺ activates Ca²⁺-dependent catabolic enzymes such as phospholipases, protein kinases, proteases, and caspase cascades (White et al, 2000), which subsequently induce neuron necrosis and apoptosis. A number of calcium channel blockers and N-methyl-d-aspartate (NMDA)/receptor antagonists have been developed and tested for ischemic stroke treatment (Asakura et al, 1997; Kobayashi and Mori, 1998; Kume et al, 2002; Perez-Pinzon et al, 1997; Zipfel et al, 1999); however, most of these drugs have failed in clinical trials because of either intolerable side effects or lack of therapeutic efficacy (Lee et al, 1999).

Calbindin D 28k (CaBD) is a member of the superfamily of calcium-binding proteins. It is found in neurons and astrocytes throughout the nervous system (Baimbridge et al, 1992). Physiologically, CaBD can buffer and modulate intracellular calcium concentration ([Ca²⁺]_i) to avoid neuronal injury; however, the buffering ability is greatly discounted in ischemic injury because of the large increase of [Ca²⁺]_i. It has been reported that exogenous CaBD can reduce oxidative stress, preserve mitochondrial function (Guo et al, 1998), and protect neurons against glutamate- or ischemia-induced delayed excitotoxicity (D'Orlando et al, 2001, 2002; Meier et al, 1998). Therefore, increasing intracellular CaBD expression and buffering the excessive Ca²⁺ might provide a unique approach to reduce [Ca²⁺]_i cytotoxicity.

In the present study, we hypothesized that buffering the excessive Ca²⁺ in the cytosol using CaBD, rather than blocking Ca²⁺ influx, would provide neuroprotection during cerebral ischemia. We firstly generated a novel fusion protein PTD-CaBD (PTD, protein transduction domain) in Escherichia coli; then, we further examined (1) whether PTD-CaBD could cross the cell membrane into cells; (2) whether the PTD-CaBD had biologic activity ex vivo; and (3) whether PTD-CaBD attenuated ischemic brain injury in the rat transient middle cerebral artery occlusion (tMCAO) model in vivo.

Materials and methods

Generation of Recombinant Fusion Proteins

The PTD-CaBD was generated according to the method described previously (Fan et al, 2006). Calbindin D 28k encoding sequence was PCR amplified using the primers 5′ ata gga tcc gca gaa tcc cac ctg cag tc 3′ (sense, modified by BamHI at 5′) and 5′ ata aga ata agc ggc cgc cta gtt gtc ccc agc aga gag aa 3′ (antisense, modified by NotI at 3′) from rat brain cDNA. The purified PCR fragment was cloned into the BamHI/NotI site of the pTransvector (qBiogene, St Louis, MO, USA), and into the pET32a (Novagen, Madison, WI, USA) as a control. PTD-CaBD and thioredoxin (Trx)-CaBD were expressed in E coli BL21 (DE3). The fusion proteins were extracted and purified under native conditions. Briefly, the bacteria pellet was suspended in a lysis buffer (50 mmol/L NaH₂PO₄, 500 mmol/L NaCl, 10% glycerol, 10 mmol/L imidazole and 1 mmol/L PMSF, pH 8.0). After ultrasonication and centrifugation, the supernatant was subjected to metal-affinity chromatography using a Ni-NTA resin column (Novagen). The column was washed with 20 mmol/L imidazole and finally eluted in elute buffer (50 mmol/L NaH₂PO₄, 300 mmol/L NaCl, and 500 mmol/L imidazole, pH 8.0). Finally, excessive salt and imidazole were removed from the purified product through dialysis using the 7,000 MWCO dialysis cassettes (Pierce, Rockford, IL, USA) in phosphate-buffered saline (PBS).

Some of the two recombinant proteins were labeled with fluorescein isothiocyanate (FITC) (Mao, 1994). Briefly, the protein solution was dialyzed overnight in 500 mmol/L carbonate, pH 9.5. Then, FITC dissolved in dimethyl sulfoxide (10 mg/mL) was added at a ratio of 40 to 80 μg FITC per mg protein. The solution was incubated with gentle shaking at room temperature for 1 h. Free FITC was removed thoroughly by dialysis in PBS. The PBS from the last time dialysis was collected and used as a control as described below.

PTD-CaBD Transduction

Cos7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Invitrogen, San Diego, CA, USA). To observe the transduction ability, Cos7 cells were cultured up to 75% confluence and incubated with 1.0 μmol/L PTD-CaBD or Trx-PTD for 1 h at 37°C. The cells were washed and images were taken through a fluorescent microscope (Olympus, Melville, NY, USA). The time course and dose-dependent analysis were performed using flow cytometry. Different concentrations of PTD-CaBD (0 to 2.0 μmol/L) were used for the dose-dependent study, and time points of 0 to 120 mins were used for the time-course study. The cells were washed, trypsinized, and resuspended in PBS at 1 × 10⁶ cells/mL for flow cytometry.

Hippocampal Slice Culture

The institutional animal care and use committee at Shanghai Medical School, Fudan University, China approved all procedures for the use of laboratory animals. Hippocampal slices were prepared according to the method of Stoppini et al (1991). Briefly, hippocampus was isolated on day 1 from a neonatal Sprague—Dawley rat. Transverse hippocampal slices of 400 μm in thickness were planted on six-well Transwell insert membranes (Corning Costar, Cambridge, MA, USA) and cultured in Neuro-basal culture medium (Invitrogen). To determine PTD-CaBD transduction efficiency, 2 μmol/L of PTD-CaBD-FITC or Trx-CaBD-FITC was added into the culture media. The plates were washed after 1 h of incubation. Images were captured using an inverted fluorescent microscope.

NMDA and OGD Exposure of Hippocampal Slices

Hippocampal slices exposed to NMDA and oxygen—glucose deprivation (OGD) were used to evaluate the biologic activity of PTD-CaBD. The hippocampal slices were incubated in a medium with 100 μmol/L NMDA (Sigma, St Louis, MO, USA) for 1 h at 37°C to induce Ca²⁺ influx. For the OGD treatment, inserts with slices were washed and transferred into neurobasal culture medium without glucose (Invitrogen) in six-well plates. The plates were put into an airtight microincubator chamber that was sealed tightly after being flushed with 95% N₂/5% CO₂ for 10 mins. The slices in the tightly sealed chamber were incubated at 37°C for 20 mins and then submerged into normal culture condition.

Ca²⁺ Assay

[Ca²⁺]_i in hippocampal cells was measured using the sensitive indicator Fluo 3-AM (Yang and Steele, 2005). Hippocampal slices were loaded with Fluo 3-AM (5 μmol/L) in D-Hanks and incubated at 37°C for 60 mins. The slices were washed to remove free Fluo 3 AM and incubated in a serum-free medium. A confocal microscope (Bio-Rad, Hercules, CA, USA, Microradiance 2000) was used to detect [Ca²⁺]_i.

Cell Death Determination

Propidium iodide (PI, 2 μg/mL) was added to the culture medium before or after treatment. Propidium iodide fluorescence images were captured from each slice immediately after PI application as the baseline measurement (F_b). Additional measurements were performed at different time points after treatment (F_t). At the end of the experiment, the slices were lethally challenged with NMDA and Kainic acid (KA) (500 μmol/L in PBS) for 2 h at 37°C and 24 h at 4°C. Additional PI fluorescence images were taken as F_max. The percentage of cell death in each slice was expressed as Death% = (F_t–F_b)/(F_max–F_b) × 100%, where F_t is slice fluorescence at time t, F_b is the background florescence at the start of the experiment, and F_max was the maximal fluorescence after complete cell killing (Abdel-Hamid and Tymianski, 1997).

Transduction of PTD-CaBD in the Brain

Adult male Kunming mice were anesthetized with 1.5% isoflurane, and then PTD-CaBD (50 μg/kg) or Trx-CaBD (50 μg/kg) was intraperitoneally injected into mice. Animals were killed 8 h after injection and the brains were perfused transcardially with heparinized saline followed by 4% paraformaldehyde/PBS. Brains were removed and postfixed in 4% paraformaldehyde/PBS overnight at 4°C. Frozen sections 10 μm in thickness were prepared for immunoassaying. Anti-polyhistidine antibody (1:500 dilution; Novagen) and FITC-conjugated IgG (1:2,000 dilution; Sigma) were used to detect the histidine-tagged PTD-CaBD.

tMCAO in Rats

Adult male Sprague—Dawley rats weighing 200 to 220 g were anesthetized with 1.5% isoflurance. The internal carotid artery was isolated and the pterygopalatine artery was ligated. Then, a 3 cm length of 4-0 nylon suture with a slightly larger tip was gently advanced from the external carotid artery to the beginning of the middle cerebral artery for a distance of 18.0 ± 0.5 mm. The suture was withdrawn from the internal carotid artery to the common carotid artery for reperfusion (Longa et al, 1989; Zhang et al, 2003). The animals underwent 60 mins of tMCAO and 72 h of reperfusion. Surface cerebral blood flow was measured before and during MCAO using laser-Doppler flowmetry (Peri Flux System 5000, Perimed, Sweden) to ensure the success of MCAO. The rats were divided into the following experimental groups (six rats per group): (1) a sham control group; (2) tMCAO rats with saline intraperitoneal injection; and (3) rats with PTD-CaBD intraperitoneal injection (5 μg/kg) 30 mins before tMCAO.

Neurologic Severity Scores

Modified neurologic severity scores were used to evaluate neurologic function of each animal (Chen et al, 2001); neurologic severity score is a composite of motor, sensory, reflex, and balance tests. Neurologic function was graded on a scale of 0 to 18 (normal, 0; maximal deficit score, 18). The rat was scored 0 if it could pass all the tests, and the higher the score, the more severe the injury. All animals were trained to be familiar with the testing environment before the MCAO surgery. Neurologic examination was conducted in surviving animals at 24 and 72 h by a maskedinvestigator.

Infarct Area Determination

TTC (2,3,5-triphenyl-tetrazolium chloride; Sigma) staining was performed to detect the infarct volume according to Wang et al (2004). The brains were removed 72 h after reperfusion, and a series of 2 mm coronal brain slices were stained in 1% TTC in 0.1 mol/L PBS for 30 mins at 37°C. Infarct volumes were measured by sampling stained sections with a digital camera (Nikon Corporation, Tokyo, Japan). The ischemic area was calculated as the difference between the area of the nonischemic hemisphere and the normal area of the ischemic hemisphere. The infarct volume was calculated by multiplying the infarct areas by the thickness of sections (Cao et al, 2002).

TUNEL Staining

The terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay kit (Oncogene, Boston, MA, USA) was used to detect DNA fragmentation. The brain sections were fixed in 4% paraformaldehyde/PBS for 15 mins, and incubated in 20 μg/ml proteinase K/PBS at room temperature for 10 mins. Then, 3% H₂O₂ was used to quench the endogenous horseradish peroxidase. The sections were then incubated in the labeling reaction mixture for 90 mins at 37°C. Anti-BrdU-biotin (1:20 dilution) was applied for 60 mins. After incubating in conjugate buffer, sections were stained with DAB-H₂O₂ (DAB, diaminobenzidine tetrahydrochloride), and counterstained with Harri's hematoxylin.

Immunostaining

The frozen brain sections were incubated in 1% Triton X-100 for 15 mins, in 3% H₂O₂ for 5 mins, and then blocked using 1% bovine serum albumin (Sigma) for 30 mins. The sections were incubated in primary antibody (anti-cleaved caspase-3 antibody, 1:100 dilution, Cell Signaling Tech, Beverly, MA, USA; anti-polyhistidine antibody, 1:500 dilution, Novagen) overnight at 4°C and in FITC- or horseradish peroxidase-coupled IgG antibody (1:100 dilution; Hua-Mei Biotech, Shanghai, China) for 1 h at room temperature. Finally, the sections were detected by fluorescent microscopy or by using a DAB reagent (Sigma).

To analyze TUNEL and cleaved caspase-3-positive staining, two coronal sections (20 μm thickness) 2 mm anterior and 1 mm posterior to the Bregma were chosen. We randomly selected five fields of view around the boundary zone of the ischemic core in each section. We then counted the total number of positive cells in each field. Finally, we calculated the average cell number per field of view. The average was used to deduce positive cells in the whole boundary zone.

Western Blot

Protein was extracted from the ischemic zone and the concentration was determined using bicinchoninic acid assay. An equal volume of proteins (80 μg) with sample buffer was boiled and loaded onto 15% polyacrylamide gels. Proteins were transferred onto a Hybond N nylon membrane (Amersham, Piscataway, NJ, USA). The membrane was incubated in 5% milk/PBS blocking solution, and immersed in anti-cleaved caspase-3 (1:250 dilution; Cell Signaling) or anti-polyhistidine antibody (1:500 dilution; Novagen) overnight. Then horseradish peroxidase-labeled anti-rabbit second antibody (1:100 dilution; Hua-Mei Biotech) was used to detect the immunoreactivity. Lastly, the membrane was developed in color with an enhanced ECL kit (Amersham) and exposed to Kodak film.

Statistical Analysis

All data were presented as mean ± standard deviation (s.d.). Student's t-test was used to compare the difference between the treated or untreated group. A probability of less than 1% or 5% was considered to be statistically significant.

Results

PTD-CaBD Generation

The fusion proteins were tagged 6 histidines for purification (Figure 1A). To generate the proteins, full-length CaBD-encoding cDNA was amplified. The fragment was purified by gel extraction and subsequently was cloned into pTransVector containing the PTD peptide, and into pET32a containing Trx but not PTD as a control. The recombinant plasmids pTV-CaBD and pET32-CaBD were verified by enzyme digestion and PCR amplification, indicating the insert of 786 bp fragments (Figure 1B).

Figure 1

Generation of the fusion proteins PTD-CaBD and Trx-CaBD. (A) Structure of the PTD-CaBD and Trx-CaBD. (B) Constructing the expression plasmids. Lane 1 and 2, recombinant plasmids pTransvecter-CaBD and pET32a-CaBD releasing the insert of 786 bp after being enzyme-digested; lanes 3 and 4, PCR products of CaBD amplified from the two recombinant plasmids. (C) Coomassie brilliant blue staining of the expression of the fusion proteins in E coli BL21. Lane 1, a control without plasmid transduction; lanes 2 and 4, BL21 transfected with pTransvector-CaBD or pET32a-CaBD; lanes 3 and 5, PTD-CaBD and Trx-CaBD after purification by Ni-NTA resin columns. (D) Western blotting verification of PTD-CaBD and Trx-CaBD. Lane 1, PTD-CaBD, 28 kDa; lane 2, Trx-CaBD, 45 kDa, containing the Trx protein.

The sequencing analysis showed that the fragment matched exactly with rat brain CaBD cDNA (GI: 203236). Finally, the proteins PTD-CaBD and Trx-CaBD were expressed in E coli and then purified to near homogeneity. The fusion proteins were assayed by SDS—polyacrylamide gel electrophoresis after Coomassie brilliant blue staining and further identified using Western blot analysis; the results showed that Trx-CaBD was 45 kDa and PTD-CaBD was 28 kDa (Figures 1C and 1D).

Transduction Efficiency of PTD-CaBD

PTD-CaBD-FITC was efficiently delivered into cos7 cells after 1 h of incubation, whereas Trx-CaBD-FITC was not detected in cos7 cells (Figure 2A). Flow cytometer assay showed that PTD-CaBD was delivered into cos7 cells in a dose-dependent manner, and 0.05 μmol/L of FITC-PTD-CaBD could be delivered into approximately 80% cells (Figure 2B). FITC-PTD-CaBD-positive cells increased significantly after 10 mins of incubation, and peaked at 1 h (Figure 2C).

Figure 2

PTD-CaBD could be delivered into cos7 cells efficiently. (A) Photographs showing cos7 cells incubated with Trx-CaBD-FITC (a) or PTD-CaBD-FITC (b); fluorescence-positive staining was detected in PTD-CaBD- but not in Trx-CaBD-transfected cos7 cells. The delivered fusion protein mainly distributed in the cytoplasm; bar = 20 μm. (B) The bar graph showing the dose-dependent pattern of PTD-CaBD transfection. All the experiments were repeated three times. (C) The line graph showing the time course of PTD-CaBD transfection, indicating that more than 70% of cos7 cells were PTD-CaBD-FITC positive after 10 mins of incubation.

Effect of PTD-CaBD on OGD or NMDA Exposure

Most cells in PTD-CaBD-FITC-treated slices showed strong fluorescence-positive staining after 1 h of incubation, whereas cells in Trx-CaBD-FITC-treated slices showed negative staining (Figure 3A). After pretreatment with 0.1 μmol/L PTD-CaBD for 1 h, intercellular Ca²⁺ in hippocampal slices subjected to OGD or 100 μmol/L NMDA significantly decreased compared with the control groups (P < 0.05; Figures 3B and 3C). Further analysis showed that pretreatment with different concentrations of PTD-CaBD (0, 10, 50, and 100 nmol/L) could attenuate hippocampal slice cell death induced by NMDA in a dose-dependent manner (Figures 3D and 3E).

Figure 3

PTD-CaBD could buffer excessive Ca²⁺ in hippocampal slice cells. (A) Photographs showing strong fluorescence-positive staining in PTD-CaBD-FITC-transfected hippocampal cells (b), but not in Trx-CaBD-FITC-transfected hippocampal cells (a). Bar = 200 μm. (B) Photomicrographs showing Fluo3 AM-positive staining in the hippocampal slice cells, using a confocal microscopy. (a) A hippocampal slice treated with NMDA for 1 h; (b) a hippocampal slice pretreated with 0.1 μmol/L PTD-CaBD after NMDA treatment; (c) a hippocampal slice subjected to 20 mins of OGD; (d) a hippocampal slice pretreated with 0.1 μm PTD-CaBD after OGD treatment. Hippocampal slices treated with NMDA and OGD show fluorescence-positive staining, whereas PTD-CaBD-treated hippocampal slice are negative. Bar = 200 μm. (C) Bar graph showing statistical analysis of (B). Data are mean ± s.d.; n = 3 in each group, *P < 0.05 compared with the non-pretreated group. (D) Photographs showing Pl-positive staining in the NMDA-treated hippocampal slices with different concentrations of PTD-CaBD (0, 10, 50, and 100 nmol/L). Bar = 200 μm. (E) Bar graph showing the % of maximum cell death in the NMDA-treated hippocampal slices with different concentrations of PTD-CaBD. Data are mean ± s.d.; N = 3 in each group; *P < 0.05, versus untreated slices.

Effect of PTD-CaBD on Rat Brains after tMCAO

The surface cerebral blood flow was measured before and during MCAO operation to ensure the success of the model. The surface cerebral blood flow in the ipsilateral hemisphere of rats with tMCAO was reduced to 10% to 20% of baseline. The surface cerebral blood flow returned to at least 80% within 20 mins after the thread was withdrawn. There was no difference in physiologic parameters (e.g., arterial blood pressure, blood gases, body temperature) during tMCAO among the different experimental groups (data not shown).

Immunofluorescence staining after 8 h of PTD-CaBD injection showed strong positive polyhistidine immunoreactivity in the subventricular zone, cerebral cortex, and caudate putamen injected with PTD-CaBD (Figure 4A). Interestingly, we did not find any detectable differences in the distribution of PTD-CaBD between the control group and PTD-CaBD injection plus MCAO group (data not shown), which indicated that PTD-mediated protein delivery into brain was not because of blood—brain barrier breakdown induced by ischemia. Infarct volume of PTD-CaBD-treated rats was significantly smaller than that of the control group (280 ± 47 versus 166 ± 70 mm³ P < 0.05; Figures 4B and 4C). Furthermore, behavioral outcome evaluated by neurologic severity scores had better scores in the PTD-CaBD-pretreated rat after 72 h of reperfusion compared with the controls (P < 0.05; Figure 4D).

Figure 4

PTD-CaBD reduced brain infarct volume and improved neurologic outcomes in tMCAO rats. (A) Photomicrographs showing histidine tag immunofluorescence in brains injected with Trx-CaBD (a) or PTD-CaBD (b—d). A number of positive cells appear in the subventricular zone (b), caudate putaman (c), and cerebral cortex (d) in PTD-CaBD-treated mouse brains whereas the histidine tag shows negative in Trx-CaBD-treated brains. V, lateral ventricle; CP, caudate putaman; CTX, cerebral cortex. Bar = 50 μm. (B) Photographs showing the infarct area detected by TTC staining in tMCAO (left panel) and tMCAO pretreated with PTD-CaBD (right panel), indicating smaller infarction area in the PTD-CaBD group. (C) Bar graph showing the infarct volume measurement in tMCAO rats pretreated with or without PTD-CaBD. Data are mean ± s.d.; N = 6 in each group; P < 0.05, compared with the untreated tMCAO group. (D) Bar graph showing the changes of neurologic severity score (NSS) in tMCAO rats pretreated with or without PTD-CaBD after 24 and 72 h of reperfusion. Data are mean ± s.d.; N = 6 in each group; *P < 0.05, compared with the score after 24 h reperfusion.

The Action of PTD-CaBD in Apoptosis Inhibition

TUNEL-positive cells in the ischemic boundary zone increased after ischemia, but were significantly reduced in the PTD-CaBD-treated group compared with the untreated group (58% ± 7% versus 29% ± 3%, P < 0.05; Figures 5A and 5C). Similarly, the cleaved caspase-3-positive cells significantly increased after ischemia/reperfusion compared with the controls. The positive cells were detected mainly in the ischemic perifocal region, although there were a few positive cells in the ischemic core areas. Importantly, the cleaved caspase-3-positive cells significantly decreased in the PTD-CaBD-treated rats compared with the tMCAO group (62 ± 4/field versus 31 ± 6/field, P < 0.05; Figures 5B and 5D). The Western blot result was parallel to the immunostaining findings (P < 0.05; Figures 5E and 5F).

Figure 5

PTD-CaBD inhibited brain cell apoptosis. (A) Photomicrographs showing TUNEL-positive cells in the sham group (a), tMCAO group (b), and tMCAO pretreated with PTD-CaBD group (c). No TUNEL-positive staining was detected in the sham animals. Many TUNEL-positive cells were detected in the ischemic boundary zone in the tMCAO rats, whereasTUNEL-positive cells are much less in the PTD-CaBD-pretreated tMCAO rats. Bar = 50 μm. (B) Photomicrographs showing the immunostaining of cleaved caspase-3 in the sham group (a), tMCAO group (b), and tMCAO pretreated with PTD-CaBD group (c). Cleaved caspase-3-positive cells were scarcely distributed in the brain sections of sham group (a). Many cleaved caspase-3-positive cells are detected after tMCAO (b). Cleaved caspase-3-positive cells are attenuated in the rats pretreated with PTD-CaBD (c). Bar = 50 μm. (C) Bar graph showing the quantitative statistical analysis of the percentage of TUNEL-positive cells. Data are mean ± s.d., N = 6 per group. *P < 0.05, tMCAO with PTD-CaBD treatment versus tMCAO alone. (D) The bar graph showing the quantitative analysis of the cleaved caspase-3-positive cell number in each group. Data are mean ± s.d., N = 6 per group. *P < 0.05, tMCAO with PTD-CaBD treatment versus tMCAO alone.

Discussion

For the first time, we used PTD-mediated protein transduction to explore the effect of CaBD in focal cerebral ischemic animal models. The results show that (1) PTD-CaBD could be delivered into cos7 cells or brain slices, and they buffer the excessive calcium, and therefore reduce NMDA- and OGD-induced neuronal injury, and (2) PTD-CaBD could be delivered into the rat brain and thus protect it from ischemia/reperfusion injury.

Protein transduction domains, also termed cell-permeable proteins or membrane translocating sequences, are small peptides that are able to ferry much larger molecules into cells independent of classical endocytosis (Beerens et al, 2003; Joliot and Prochiantz, 2004). This provides an exciting therapeutic opportunity for the treatment of brain ischemia and many other diseases. The PTD peptides can be found in several proteins such as TAT protein of the human immunodeficiency virus (HIV-tat) and antennapedia of Drosophila (Antp HD) (Leifert et al, 2002). Delivery of proteins or peptides fused to PTD into many kinds of cells in vitro or in vivo, even across the blood—brain barrier into brain parenchyma, has been reported (Dietz and Bahr, 2004). These proteins maintain their biologic activity and exert therapeutic action. Our previous studies showed that XIAP fused with PTD could inhibit apoptosis in the ischemic brain (Fan et al, 2006).

In our experiments, we found that 70% to 80% of cos7 cells were transduced even at a very low concentration of PTD-CaBD (0.05 μmol/L), or after a short incubation time (begins at 10 mins and peaks at 1 h), suggesting that PTD-mediated protein transduction was rapid and efficient. Moreover, the transduction could occur at 4°C and in the presence of known endocytosis inhibitors. The mechanism of PTD-mediated protein transduction, independent of energy and receptors binding, has not yet been fully elucidated. A nonspecific electrostatic interaction of basic amino acids PTD with cellular membrane might be the first crucial step (Bellet-Amalric et al, 2000); heparan sulfate in the cytoplasm (Rusnati et al, 1999; Ziegler and Seelig, 2004) and amphiphilic α-helices of PTD might be required during the transduction (Scheller et al, 1999). For the hippocampal slices, we obtained a similar result; after incubation with FITC-labeled PTD-CaBD, most of the cells showed FITC—positive staining.

After delivery into cells, the PTD fusion proteins in a partially denatured state are refolded and/or modified to perform their biologic functions (Schneider et al, 1996; Schwarze et al, 1999). In our experiments, when the hippocampal slices pretreated with PTD-CaBD were exposed to NMDA or OGD, [Ca²⁺]_i significantly decreased and the cell death was much less than the control group. These results suggest that PTD-CaBD was not only delivered into cell cytosol, but also performed its biologic action through binding the overloaded [Ca²⁺]_i and blocking the downstream lethal cascade, and consequently protecting the cells from OGD- or NMDA-induced injury. Previous reports also indicated that overexpression of CaBD protected glutamate-induced excitotoxicity in U937 cells (Jeon et al, 2004), neuroblastoma—retinal hybrid cells (D'Orlando et al, 2002), and pancreatic islet beta cells (Parkash et al, 2002; Rabinovitch et al, 2001).

Calbindin D 28k is a well-characterized Ca²⁺-binding protein; its major role is to buffer and transport Ca²⁺ to maintain calcium homeostasis (Heizmann and Braun, 1992). Expression of CaBD increases in many central nervous system diseases, such as Alzheimer's disease (Geula et al, 2003), transient forebrain ischemia (Hwang et al, 2003), and spinal ischemia (Lee et al, 2005), which may be associated with resistance to neuronal degeneration. Overexpression of CaBD could decrease neuronal death after transient focal ischemia (Yenari et al, 2001). In contrast to the low transfection, PTD can deliver its cargo into brain cells more efficiently across the blood—brain barrier into neurons and astrocytes in the cerebral cortex, striatum, and medulla oblongata (Asoh et al, 2005; Cao et al, 2002; Kilic et al, 2003). After injection of 50 μg/mL PTD-CaBD, we detected a number of anti-histidine-positive cells in the subventricular zone, cerebral cortex, and caudate putamen. The high efficiency of protein transduction makes it possible to apply systemically PTD fusion proteins in stroke therapy. Actually, we found that the infarct volume decreased and the neurologic recovery improved in the PTD-CaBD-treated rats compared with the non-treated control group. Although the neuroprotective effect of CaBDs appears controversial (Airaksinen et al, 1997; Bouilleret et al, 2000; Kiyama et al, 1990; Simic et al, 1999), our data indicate that the PTD-CaBD could be delivered into brain cells across the blood—brain barrier, and thus buffer ischemia-induced Ca²⁺ overload and attenuate the brain injury. Admittedly, organ-specific delivery remains a complex challenge with respect to the utility of this technology (Noguchi and Matsumoto, 2006) because systemic administration of PTD fusion proteins can be distributed in the liver, kidney, spleen, lung, bowel, and heart (Cai et al, 2006), which might cause unexpected side effects. Interestingly, in our experiments, we did not find detectable side effects after PTD-CaBD injection. This may result from the fact that the exogenous CaBD does not block the calcium channels but buffers the excessive intracellular calcium; therefore, normal calcium influx maintains the physiologic calcium homeostasis to avoid cell injury.

Brain ischemia not only leads to acute cell death in the ischemic core, but also initiates cell apoptosis, which is the major component of delayed neuronal injury in the boundary area (Kametsu et al, 2003; Nitatori et al, 1995). Ca²⁺ overload in mitochondria induced by ischemia activates caspase-9, which subsequently activates caspase-3, initiating the mitochondria-dependent apoptosis pathway (Chen et al, 2005; Miyamoto et al, 2005). Our results showed that delivery of PTD-CaBD significantly decreased TUNEL- and cleaved caspase-3-positive cells in the perifocal region, which suggests that PTD-CaBD could inhibit ischemia-induced apoptosis. This antiapoptotic effect results not only from its ability to chelate calcium and therefore inhibit caspase-9 activation, but also from its ability to directly bind and inhibit caspase-3 activity (Bellido et al, 2000).

Footnotes

Acknowledgements

We thank Huimin Ren, PhD, and neuroscientist in the Fudan University for experiment assistance. We also thank Jiahong Lu PhD, MD, and Qiang Dong, PhD, MD, Neurologists in the Fudan University, for the useful suggestions about the project.

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Pretreatment with PTD-Calbindin D 28k Alleviates Rat Brain Injury Induced by Ischemia and Reperfusion

Abstract

Keywords

Introduction

Materials and methods

Generation of Recombinant Fusion Proteins

PTD-CaBD Transduction

Hippocampal Slice Culture

NMDA and OGD Exposure of Hippocampal Slices

Ca2+ Assay

Cell Death Determination

Transduction of PTD-CaBD in the Brain

tMCAO in Rats

Neurologic Severity Scores

Infarct Area Determination

TUNEL Staining

Immunostaining

Western Blot

Statistical Analysis

Results

PTD-CaBD Generation

Transduction Efficiency of PTD-CaBD

Effect of PTD-CaBD on OGD or NMDA Exposure

Effect of PTD-CaBD on Rat Brains after tMCAO

The Action of PTD-CaBD in Apoptosis Inhibition

Discussion

Footnotes

Acknowledgements

References

Ca²⁺ Assay