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Analysis of a new gene related to low K+-induced apoptosis of cerebellar granule neurons
s333
769
ANALYSIS
OF A NEW GENE RELATED
GRANULE
NEURONS
KIYOMI YOSHIDA,
APOPTOSIS
OF CEREBELLAR
CHIKA NISHIO, HIROSHI HATANAKA
Division of Biosynthesis, After cerebellar
TO LOW K+-INDUCED
Institute for Protein Research, Osaka University,
granule neurons from 1 l-day-old
Suita, Osaka 565-087 I, Japan
3-2 Yamadaoka,
rats were cultured under the high potassium
medium for 4 days, cells were changed to the low concentration
of potassium
die. This low K’ -induced cell death is thought to be apoptosis because DNA fragmentation was blocked by Actinomycin
D, RNA synthesis inhibitor.
concentration
We cultured cerebellar
was observed
was not induced cell death, then we cloned the subtracted RNA. hybridization
Positive clones were screened by RNase protection 4-5 kbp and was expressed
INHIBITION OF PHOSPHATIDYLINOSlTOL 3-KINASE ACTIVITY N-TERMINAL KINASE ACTIVITY IN APOPTOSIS OF CULTURED NEURONS.
SATORU YAMAGISHI, HATANAKA
KOJI SHIMOKE,
MASASHI
Inst. for Protein Research,
Osaka Univ., 3-2 Yamadaoka,
from RNA which assay.
after changing to the low K’ medium 3 hours later. Northern
results said that mRNA of this clone was approximately
770
and cell death
neurons under high K’ medium for 4 days.
and RNA was obtained from 3 hours after changing to the low K’ medium, then the RNA was subtracted We found that one of the clones increased the RNA expression
(26 mM)
(5 mM) medium, the most cells were going to
YAMADA,
TOSHIHIKO
especially
in neurons.
ELEVATES C-JUN CEREBELLAR GRANULE IKEUCHI
and HIROSHI
Suita, Osaka 565.087~
Cerebellar granule neurons maintained in medium containing 26 mM potassium or in medium (5 mM potassium) with 50 q/ml BDNF undergo an apoptotic cell death vvhen exposed to 10 ,1 M LY294002, an inhibitor of phosphatidyiinositol 3-kinase (PI3-K). This cell death was blocked by 0.3 ,1 M cycloheximide or 0.1 fl M actinomycin D, suggesting that dr ~io\~o RNA and protein synthesis are required. To investigate the intracellular signaling mechanism of LY294002-induced apoptosis, the activity of Akt was measured in cells in HK’ (26 mM potassium) medium or LK’ (5 mM potassium) medium containing BDNF, with or without 10 /L M LY294002. Akt activity decreased following the addition of 10 fl M LY294002. Similar results were obtained with 100 nM wortmannin, another inhibitor of PI3-K. In addition, we found that LY294002 increased the c-Jun N-teminal kinase (JNK) activity, which is believed to mediate many types of neuronal cell death. We also observed elevated expression of c-Jun by LY294002 in HK’ or LK’+ BDNF. These findings demonstrated that apoptosis induced by inhibition of PI3K activity involves suppression of the Akt activ;ity and elevation of both JNK activity and c-Jun expression. Our results suggested that the PI3-K-Akt pathway suppresses the activation of JNK and c-Jut1 expression, and as a result prevents the neuronal cell death in cerebellar granule neurons.
771
RELATIONSHIP
HIDETOSHI
IN0 AND TANEMICHI
CELL-CYCLE
Third Department
BETWEEN REGULATORY
THE NEURONAL
CELL DEATH AND THE EXPRESSION
CHIBA
of Anatomy, Chiba University
School of Medicine,
Several lines of evidence suggest that there is relationship
1-8-1 Inohana, Chuo-ku. Chiba 260-8670
between the induction of apoptosis and the expression
cell-cycle regulatory proteins. Especially in the nervous system, it has been reported that the up-regulation expression
is accompanied
by the neuronal apoptosis. We investigated
regulatory proteins in rat brains systemically
the alteration of the expression
injected with kainate, using in situ hybridization.
of CDK4, cyclin D 1 and cyclin D2 was observed in neurons surrounding
suggest that the CDK4-cyclin
of cell-cycle
Kainate induced the of the
the apoptotic areas. These data
D system acts an important role in the induction of neuronal apoptosis.
of
of the cyclin Dl
neuronal apoptosis in the amygdala and piriform cortex 2 days after kainate injection. Significant up-regulation expression
OF THE
PROTEINS.
769
ANALYSIS
OF A NEW GENE RELATED
GRANULE
NEURONS
KIYOMI YOSHIDA,
APOPTOSIS
OF CEREBELLAR
CHIKA NISHIO, HIROSHI HATANAKA
Division of Biosynthesis, After cerebellar
TO LOW K+-INDUCED
Institute for Protein Research, Osaka University,
granule neurons from 1 l-day-old
Suita, Osaka 565-087 I, Japan
3-2 Yamadaoka,
rats were cultured under the high potassium
medium for 4 days, cells were changed to the low concentration
of potassium
die. This low K’ -induced cell death is thought to be apoptosis because DNA fragmentation was blocked by Actinomycin
D, RNA synthesis inhibitor.
concentration
We cultured cerebellar
was observed
was not induced cell death, then we cloned the subtracted RNA. hybridization
Positive clones were screened by RNase protection 4-5 kbp and was expressed
INHIBITION OF PHOSPHATIDYLINOSlTOL 3-KINASE ACTIVITY N-TERMINAL KINASE ACTIVITY IN APOPTOSIS OF CULTURED NEURONS.
SATORU YAMAGISHI, HATANAKA
KOJI SHIMOKE,
MASASHI
Inst. for Protein Research,
Osaka Univ., 3-2 Yamadaoka,
from RNA which assay.
after changing to the low K’ medium 3 hours later. Northern
results said that mRNA of this clone was approximately
770
and cell death
neurons under high K’ medium for 4 days.
and RNA was obtained from 3 hours after changing to the low K’ medium, then the RNA was subtracted We found that one of the clones increased the RNA expression
(26 mM)
(5 mM) medium, the most cells were going to
YAMADA,
TOSHIHIKO
especially
in neurons.
ELEVATES C-JUN CEREBELLAR GRANULE IKEUCHI
and HIROSHI
Suita, Osaka 565.087~
Cerebellar granule neurons maintained in medium containing 26 mM potassium or in medium (5 mM potassium) with 50 q/ml BDNF undergo an apoptotic cell death vvhen exposed to 10 ,1 M LY294002, an inhibitor of phosphatidyiinositol 3-kinase (PI3-K). This cell death was blocked by 0.3 ,1 M cycloheximide or 0.1 fl M actinomycin D, suggesting that dr ~io\~o RNA and protein synthesis are required. To investigate the intracellular signaling mechanism of LY294002-induced apoptosis, the activity of Akt was measured in cells in HK’ (26 mM potassium) medium or LK’ (5 mM potassium) medium containing BDNF, with or without 10 /L M LY294002. Akt activity decreased following the addition of 10 fl M LY294002. Similar results were obtained with 100 nM wortmannin, another inhibitor of PI3-K. In addition, we found that LY294002 increased the c-Jun N-teminal kinase (JNK) activity, which is believed to mediate many types of neuronal cell death. We also observed elevated expression of c-Jun by LY294002 in HK’ or LK’+ BDNF. These findings demonstrated that apoptosis induced by inhibition of PI3K activity involves suppression of the Akt activ;ity and elevation of both JNK activity and c-Jun expression. Our results suggested that the PI3-K-Akt pathway suppresses the activation of JNK and c-Jut1 expression, and as a result prevents the neuronal cell death in cerebellar granule neurons.
771
RELATIONSHIP
HIDETOSHI
IN0 AND TANEMICHI
CELL-CYCLE
Third Department
BETWEEN REGULATORY
THE NEURONAL
CELL DEATH AND THE EXPRESSION
CHIBA
of Anatomy, Chiba University
School of Medicine,
Several lines of evidence suggest that there is relationship
1-8-1 Inohana, Chuo-ku. Chiba 260-8670
between the induction of apoptosis and the expression
cell-cycle regulatory proteins. Especially in the nervous system, it has been reported that the up-regulation expression
is accompanied
by the neuronal apoptosis. We investigated
regulatory proteins in rat brains systemically
the alteration of the expression
injected with kainate, using in situ hybridization.
of CDK4, cyclin D 1 and cyclin D2 was observed in neurons surrounding
suggest that the CDK4-cyclin
of cell-cycle
Kainate induced the of the
the apoptotic areas. These data
D system acts an important role in the induction of neuronal apoptosis.
of
of the cyclin Dl
neuronal apoptosis in the amygdala and piriform cortex 2 days after kainate injection. Significant up-regulation expression
OF THE
PROTEINS.