DNA fragmentation precedes aberrant expression of cell cycle-related protein in rat brain after MCA occlusion

DNA fragmentation precedes aberrant expression of cell cycle-related protein in rat brain after MCA occlusion

Hayashi, Takeshi

Recent experiments suggest that apoptotic mechanisms are involved in neuronal cell death after ischemic injury. Although the exact mechanism that triggers activation of apoptotic machinery remains uncertain, in vitro studies revealed that forced expression of cell cycle-related proteins induced apoptosis. Thus, aberrant expression of such proteins might be related to ischemic neuronal death. In the present experiment, we investigated expression of cell cycle-related proteins, i.e., cyclin B 1, cyclin Dl, cdk4, and PCNA, in rat brain after transient MCA occlusion, and compared the temporal profile of the results with that of TUNEL study, which detects double strand breaks in DNA. There were no immunoreactivities for cyclin B 1, cyclin D1, and PCNA in the brain with and without ischemia. As for cdk4, however, it became present at I and 3 days of reperfusion after 2 h of ischemia. On the other hand, TUNEL positive cells appeared as early as 3 h of reperfusion, which peaked at 1 and 3 days. These results indicate that aberrant expression of cdk4, but not cyclin B 1, cyclin D I or PCNA, actually takes place in the brain after MCA occlusion, but this is not the causative mechanism of apoptotic cell death in the brain with ischemia. [Neurol Res 1991; 21: 695-698]

Keywords: Apoptosis; brain; cell cycle; cyclin; cyclin dependent kinase; infarct

INTRODUCTION

Apoptosis is a form of cell death which occurs during physiological and some pathological conditions. In the brain with experimental ischemia, certain neuronal subpopulations die with some apoptotic features such as appearance of apoptotic bodies1, positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)2, and induction of caspases3. However, the exact mechanism which triggers activation of apoptotic machinery is yet to be resolved.

As neurons in adult brain are terminally differentiated and post-mitotic cells, they do not replicate genomic DNA. Furthermore, it is reported that forced expression of cell cycle-related proteins in these cells did not cause mitosis nor DNA replication, but caused apoptotic cell death4. Therefore, it might be possible to assume that disregulation of cell cycle-related proteins expression leads to neuronal cell death in the injured brain. In fact, in the brain with Alzheimer’s disease, there was a good correlation between the neuronal loss and presence of cell cycle markers5. However, expression of such proteins might not be the direct cause of apoptotic cell death, but only an epiphenomenon. Previous reports demonstrated that cell cycle-related proteins such as cyclin Dl and cyclin dependent kinase 4 (cdk4) are expressed in viable neurons6,7.

In the present study, we investigated expression of cell cycle-related proteins, i.e., cyclin 131, cyclin Dl, cdk4, and proliferating cell nuclear antigen (PCNA) in rat brain after transient middle cerebral artery (MCA) occlusion with immunohistochemistry. Furthermore, to investigate the relationship between aberrant expression of these proteins and apoptotic cell death, we compared the temporal profiles of the results of immunohistochemical study and that of TUNEL study, which detects double strand breaks in genomic DNA.

MATERIALS AND METHODS

Adult male Wistar rats weighing 250-280 g were used. The rats were lightly anesthetized by inhalation of a 69% (vol/vol) mixture of nitrous oxide/oxygen and 1% halothane using a face mask. A midline neck incision was made and the right common carotid artery was exposed, and then inhalation of anesthetics was stopped. When the animal began to regain consciousness, the right MCA was occluded by insertion of nylon thread through the common carotid artery, as our previous reports. During these procedures, body temperature was monitored and maintained at 37deg +/- 0.3degC with a heating pad. After 2 h of ischemic period, cerebral blood flow (CBF) was restored by removal of the nylon thread under light anesthesia with diethyl ether. Between MCA occlusion and CBF restoration and after CBF restoration, the surgical incision was closed, and the animals were allowed free access to water and food at ambient temperature (21 -23degC). This experiment was approved by the Animal Committee of Tohoku University School of Medicine.

For immunohistochemical analysis and TUNEL study, the animals were deeply anesthetized with pentobarbital (50 mg kg^sup -1^ i.p.) at 3 h, 1, 3, 7 and 14 days after CBF restoration (n=3 in each time point). The brain was perfused with heparinized physiological saline through the left cardiac ventricle at 110 mmHg pressure until colorless fluid was obtained, followed by an additional 200 ml of 4% paraformaldehyde in phosphate buffered saline (PBS). The brains were removed, immersed in 4% paraformaldehyde, and then frozen in powdered dry ice. Brain samples from two sham-control animals, which underwent exposure of right common carotid artery without MCA occlusion, were also obtained. Coronal sections of 10 pm thickness at the caudate level were cut on a cryostat at -20degC and collected on glass slides coated with poly-L-lysine.

According to our previous report, we performed immunohistochemical studies to investigate expression of four cell cycle-related proteins, i.e., cyclin 131, cyclin D1, cdk4, and PCNA. After blocking with 10% normal serum for 2 h, the slides were incubated with primary antibodies at 1 :50 dilution and 0.3% Triton-X 100 for 20 h at 4degC. The primary antibodies used were as follows: mouse monoclonal antibody against cyclin 131 (sc-245, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), mouse monoclonal antibody against cyclin DI (sc-6281, Santa Cruz Biotechnology Inc.), goat polyclonal antibody against cdk4 (sc-826, Santa Cruz Biotechnology Inc.), and mouse monoclonal antibody against PCNA (NA03-2, Calbiochem, Cambridge, MA, USA). After quenching endogenous peroxidase activity, the slides were washed and incubated for 3 h with biotinylated anti-mouse IgG (PK-6102, Vector Laboratories, Burlingame, CA, USA) for cyclin B1, cyclin Dl, and PCNA, or anti-goat IgG (7023508R, Zymed Laboratories, San Francisco, CA, USA) for cdk4, at 1 :100 dilution in PBS. Subsequently they were incubated with avidin-biotin-horseradish peroxidase complex for 30 min, and then developed using diaminobenzidine (DAB) as a color substrate. The reaction was stopped by washing them in distilled water. Sections were counterstained with methyl green. To ascertain specific bindings of the antibodies for the proteins, a set of sections were stained in a similar way without the first antibody. Furthermore, immunohistochemical study for cdk4 was also performed after absorption of the primary antibody with antigenic peptide (sc-826P, Santa Cruz Biotechnology Inc.).

For detection of double strand breaks in genomic DNA, which is one of the features seen in apoptosis, we performed TUNEL study in accordance with our previous report, with use of a kit (TACS in situ apoptosis detection kit #4810-30K, Trevigen, Gaithersburg, MD, USA)9. Briefly, the tissue sections of 10(mu)m were digested with protease K, and then incubated with terminal deoxynucleotidyl-transferase (TdT) and biotinylated dNTP for 1 h at 37degC. After washing with PBS, they were incubated with streptavidin-biotin-horseradish peroxidase complex, and developed using DAB. The sections were counterstained with methyl green.

Based on the results of the immunohistochemical analysis, we performed Western blot analysis for cdk4, in order to confirm the specific binding of the primary antibody. Briefly, the animals after 3 days of reperfusion and sham-control (n=2 in each group) were decapitated under deep anesthesia with diethyl ether, and cerebral cortical samples of the right MCA territory were quickly obtained. These tissue samples were then homogenized in a lysis buffer (NaCI 0.1 mol l^sup -1^, TrisHCI 0.01 mol l^sup -1^, pH 7.5, EDTA 1 mmol 1, and aprotinin 1 mug ml-‘). The homogenates were centrifuged at 7000 xg for 15 min at 4degC, and the supernatants were used as protein samples.

Lysates equivalent to 40 mug of protein were run on the sodium dodecyl sulfate-polyacrylamide gel, together with a size marker (RPN756, rainbow colored protein, Amersham, Arlington Heights, IL, USA). The proteins on the gel were subsequently transferred to the polyvinylidene fluoride transfer membrane (Micron Separations Inc., Westboro, MA, USA), and placed in 1% powdered milk in Tween PBS (PBS with 0.1 % Tween 20) to block non-specific binding. It was then incubated with antibody against cdk4 (sc-826, Santa Cruz Biotechnology Inc.) at 1 : 100 dilution for 20 h at 4degC. After washing in PBS, the membrane was incubated with biotinylated anti-goat IgG (7023508R, Zymed Laboratories). It was washed in PBS, and incubated with avidin-biotinhorseradish peroxidase complex (PK-6102, Vector Laboratories) for 1 h. The membrane was then developed using DAB as a color substrate. The reaction was stopped by washing it in distilled water. To ascertain specific binding of the antibody for the protein, another membrane was stained in a similar way without the first antibody.

RESULTS

There were no immunoreactivities for cyclin Bi, cyclin Dl, and PCNA in the brains with sham-operation or even with ischemic injuries (not shown). As for cdk4, no immunoreactivity was present in the sham-control brain, and still no immunoreactivity at 3 h after CBF restoration (Figure la). At 1 day after reperfusion, however, it became present in sporadic neurons in the cerebral cortex at the boundary zone of MCA (Figure 1b). In addition, neurons in the lateral part of the caudate were also stained. With higher magnification, those cells showed dense immunoreactivity in the nuclei, and weak immunoreactivity in the cytoplasm (Figure 10. The number of positively stained cells increased at 3 days (Figure Id), which decreased thereafter. Brain sections without the first antibody showed no staining. Furthermore, immunoreactivity was lost by preabsorption with antigenic peptide.

Cells with double-strand breaks in DNA are detected by TUNEL in brown. In the present study, no cells were positively labeled in the sham-control brains (Figure 2a). At 3 h after CBF restoration, however, nuclei of some neurons in the cerebral cortex and lateral part of the caudate nucleus of MCA territory were detected in brown (Figure 2b). The number of TUNEL positive neurons increased at 1 day (Figure 20 and 3 days.

Representative result of Western blot analysis is shown in Figure 3. Although immunoreactive cdk4 was not detectable in the sham-control brain by immunohistochemical analysis (Figure la), a weak band was detected in the sham-control brain lysates with Western blot analysis (Figure 3, lane C). This band became more distinct in the lysates at 3 days of CBF rerstoration (Figure 3, lane 3d). This was located at the molecular weight of 34 kD, which was compatible to that of cdk4. A blot without the first antibody showed no bands (not shown). These results indicate the specific binding of the anti-cdk4 antibody.

DISCUSSION

Neurons in adult brain are terminally differentiated and post-mitotic cells. They do not replicate genomic DNA in normal conditions, and furthermore, no proliferative disorders of neuronal cell origin have been demonstrted until now. This may be because neurons possess strict control mechanisms which prevent their re-entering into the cell cycle2,10.

These replicatively quiescent cells are considered to remain in GO phase of cell cycle, which is located upstream to the R point in G1 phase10. R point is a critical point, where cells decide whether to enter cell cycle or remain quiescent; once the cell comes over this point, they replicate DNA and result in mitosis10. Cdk4 plays a critical role for the cell to come over R point”. It phosphorylates Rb and some other 12 proteins, and consequently replicates genomic DNA . Cyclin Dl is one of the proteins which binds cdk4 and regulates its activity10,11 . This binding is necessary for cdk4 to phosphorylate its substrates. PCNA is co-immunoprecipitated with cyclin Dl, and is also thought to play an important role in DNA synthesis13. On the other hand, cyclin B1 is expressed in S to M phase, and directs transition from interphase to M phase10,14.

In normal adult human brain, no immunoreactivities for cyclin 131, cyclin DI, cdk4, or PCNA was demonstrated . In rodent brain, expression of cyclin D1 and cdk4 is still controversial; there is a report that demonstrated no mRNA for cyclin D 15, but two other reports described slight immunoreactivities for cyclin D1 and cdk4′. On the other hand, so far as we know, there has been no report which demonstrated expression of cyclin 1311 and PCNA in normal brain. We here demonstrated that no immunoreactive cyclin D1 or cdk4 was detected in control brain. The reason for the difference in results is uncertain, but it may be due to the different antibody or method we employed. In this study, cdk4 but not other proteins was induced at 1-3 days after transient MCA occlusion. This is to some extent but not completely compatible with previous reports, which demonstrated normally expressing cyclin D1 and cdk4 gained stronger immunoreactivities after kainate6 and ischemic7 injuries; our study revealed no cyclin D1 expression throughout the temporal course after ischemic injury. Although cyclin Di is necessary in normal conditions for cdk4 to exert its mitotic effect, pathological conditions may cause cdk4 expression without cyclin DI. Indeed, in human brain with Alzheimer’s disease, about 10% of neurons in the vulnerable area expressed immunoreactive cdk4, though cyclin D1 expressing cells remained less than 0.6%4.

Western blot analysis revealed that lysates from control brain also had slight immunoreactivity for cdk4 (Figure 3). This might be because Western blot analysis was more sensitive than immunohistochemical analysis, and the small amount of cdk4 was detectable only by Western blot analysis in this study. It is also possible to consider that cells in the intravascular space possessed immunoreactive cdk4.

Because inappropriate signal to reenter cell cycle causes apoptosis in neurons, we investigated whether cdk4 expression precedes fragmentation of DNA by TUNEL stud Y4,16,17. As shown in Figure 2, TUNEL positive cells appeared as early as 3 h after reperfusion and peaked at 1-3 days, which is compatible with previous reports 1,2. These results signify that DNA fragmentation occurs earlier than cdk4 expression. So we could infer that cdk4 expression is not the definitive cause of apoptotic death of neurons after ischemia. The purpose and consequence of this aberrant cdk4 expression is uncertain. This might be only an epiphenomenon. However, cdk4 is not only a mitotic kinase, but also has other functions such as repairing genomic DNA 7. Therefore, expression of cdk4 might be the cells’ effort to protect themselves against ischemic DNA breaks. Further elucidation of the functions of cdks and cyclins other than those as mitotic kinases would reveal the roles of these proteins in the brain after ischemia.

CONCLUSION

We demonstrated induction of cdk4 but not cyclin 131, cyclin Dl, or PCNA in the brain after transient MCA occlusion. Although in vitro studies reported that inappropriate expression of mitotic kinases caused apoptotic cell death, this study revealed that fragmentation of genomic DNA precedes cdk4 expression in the brain after ischemia. Thus, we could infer that cdk4 expression is not the definitive cause of apoptotic death of neurons in the brain after ischemia. Induced cdk4 might be playing some roles other than those as a mitotic kinase.

ACKNOWLEDGMENTS

This work was partly supported by Grant-in-Aid for Scientific Research (B) 09470151 from the Ministry of Education, Science and Culture of Japan, and by a grant (K. Tashiro) from the Ministry of Health and Welfare of Japan.

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Takeshi Hayashi*, Masahiro Sakurai^, Koji Abet and Yasuto Itoyama*

*Department of Neurology, ^Department of Thoracic and Cardiovascular Surgery, Tohoku University School of

Medicine, Sendai, Japan

^^Department of Neurology, Okayama University School of Medicine, Okayama, Japan

Correspondence and reprint requests to: Takeshi Hayashi, Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai, 980-8574, Japan. Accepted for publication April 1999.

Copyright Forefront Publishing Group Oct 1999

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