Cloning and expression of a rat brain interleukin-1β-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons

Binhui Ni, Xin Wu, Yansheng Du, Yuan Su, Elizabeth Hamilton-Byrd, Pamela K. Rockey, Paul Rosteck, Guy G. Poirier, Steven M. Paul

Research output: Contribution to journalArticle

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Abstract

Several members of the IL1β-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE- related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K+ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K+ to low K+ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K+ ≤90 min after switching to low K+ medium (i.e., before the commitment point for apoptosis) and partially blocked by brain- derived neurotrophic factor (BDNF), which also partially rescues granule neurons from low K+-induced apoptosis. In addition, overexpression of IRP cDNA in HeLa cells results in cell death accompanied by strong internucleosomal cleavage of DNA, a typical feature of apoptosis. Finally, we detected cleavage of the putative death substrate poly (ADP-ribose) polymerase (PARP), beginning 8 hr after changing from high K+ to low K+ medium, coinciding with the time course of induced expression of the IRP gene. Our data suggest that transcriptional activation of IRP could be one of the mechanisms involved in the apoptotic death of cerebellar granule neurons.

Original languageEnglish (US)
Pages (from-to)1561-1569
Number of pages9
JournalJournal of Neuroscience
Volume17
Issue number5
StatePublished - Mar 1 1997

Fingerprint

Caspase 1
Organism Cloning
Peptide Hydrolases
Apoptosis
Neurons
Brain
Messenger RNA
Cell Death
DNA Cleavage
Poly(ADP-ribose) Polymerases
Brain-Derived Neurotrophic Factor
HeLa Cells
Transcriptional Activation
In Situ Hybridization
Down-Regulation
Complementary DNA

Keywords

  • cerebellar granule neurons
  • CPP32/YAMA/apopain
  • ICE-related protease
  • molecular cloning
  • neuronal apoptosis

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Cloning and expression of a rat brain interleukin-1β-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons. / Ni, Binhui; Wu, Xin; Du, Yansheng; Su, Yuan; Hamilton-Byrd, Elizabeth; Rockey, Pamela K.; Rosteck, Paul; Poirier, Guy G.; Paul, Steven M.

In: Journal of Neuroscience, Vol. 17, No. 5, 01.03.1997, p. 1561-1569.

Research output: Contribution to journalArticle

Ni, Binhui ; Wu, Xin ; Du, Yansheng ; Su, Yuan ; Hamilton-Byrd, Elizabeth ; Rockey, Pamela K. ; Rosteck, Paul ; Poirier, Guy G. ; Paul, Steven M. / Cloning and expression of a rat brain interleukin-1β-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons. In: Journal of Neuroscience. 1997 ; Vol. 17, No. 5. pp. 1561-1569.
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AU - Wu, Xin

AU - Du, Yansheng

AU - Su, Yuan

AU - Hamilton-Byrd, Elizabeth

AU - Rockey, Pamela K.

AU - Rosteck, Paul

AU - Poirier, Guy G.

AU - Paul, Steven M.

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AB - Several members of the IL1β-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE- related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K+ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K+ to low K+ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K+ ≤90 min after switching to low K+ medium (i.e., before the commitment point for apoptosis) and partially blocked by brain- derived neurotrophic factor (BDNF), which also partially rescues granule neurons from low K+-induced apoptosis. In addition, overexpression of IRP cDNA in HeLa cells results in cell death accompanied by strong internucleosomal cleavage of DNA, a typical feature of apoptosis. Finally, we detected cleavage of the putative death substrate poly (ADP-ribose) polymerase (PARP), beginning 8 hr after changing from high K+ to low K+ medium, coinciding with the time course of induced expression of the IRP gene. Our data suggest that transcriptional activation of IRP could be one of the mechanisms involved in the apoptotic death of cerebellar granule neurons.

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