Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture
J. L. Bruses and G. R. Pilar Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA. 1. The relationship between cycloheximide (CHX) and RNA synthesis inhibitors on trophic-deprived neuronal survival was studied with the use of primary cultures of stage (St) 34 chick...
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| creator | Bruses, J. L Pilar, G. R |
| description | J. L. Bruses and G. R. Pilar
Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA.
1. The relationship between cycloheximide (CHX) and RNA synthesis
inhibitors on trophic-deprived neuronal survival was studied with the use
of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons,
to analyze the biological process of neuronal death caused by trophic
factor withdrawal. Tissue culture conditions were refined by characterizing
the additional medium components required to obtain 100% survival, for at
least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris,
and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly
enriched neuronal cultures almost devoid of nonneuronal cells were used. 2.
The time at which trophically deprived neurons cannot be rescued by the
addition of trophic support, "commitment point," was established to be
between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of
protein translation, reduced 3H-leucine incorporation by 90-95%, at a
concentration of 10-100 micrograms/ml. The effect of the RNA transcription
blockers actinomycin D (Act-D), alpha-amanitin, and 5.6
dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into
macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25%
by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the
3H-uridine incorporation. 4. The effect of short- and long-term incubation
with CHX on neuronal survival was analyzed. Continuous application of CHX
promoted survival for 2-3 days, but thereafter neurons died regardless of
whether CIPE was present or absent. Application of CHX for 6 h from the
onset of the culture was enough to delay the commitment point up to 24 h
after plating, and the addition of CIPE at this time maintained survival
and promoted differentiation of CHX treated neurons. 5. The RNA
transcription blockers Act-D, alpha-amanitin, and DRB were applied to both
trophically deprived and trophically supported neurons, and the survival of
each was evaluated. Neither drug was effective in supporting the survival
of trophically deprived neurons in culture, and in most cases neurons even
when cultured with CIPE died within 1-2 days in the presence of either
drug. 6. Experiments using both CHX and mRNA synthesis blockers were
performed to determine the effect of blocking mRNA transcription in
trophically deprived neurons rescued by CHX. The addition of mRNA synthesis
inhib |
| doi_str_mv | 10.1152/jn.1995.74.6.2487 |
| format | Article |
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Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA.
1. The relationship between cycloheximide (CHX) and RNA synthesis
inhibitors on trophic-deprived neuronal survival was studied with the use
of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons,
to analyze the biological process of neuronal death caused by trophic
factor withdrawal. Tissue culture conditions were refined by characterizing
the additional medium components required to obtain 100% survival, for at
least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris,
and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly
enriched neuronal cultures almost devoid of nonneuronal cells were used. 2.
The time at which trophically deprived neurons cannot be rescued by the
addition of trophic support, "commitment point," was established to be
between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of
protein translation, reduced 3H-leucine incorporation by 90-95%, at a
concentration of 10-100 micrograms/ml. The effect of the RNA transcription
blockers actinomycin D (Act-D), alpha-amanitin, and 5.6
dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into
macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25%
by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the
3H-uridine incorporation. 4. The effect of short- and long-term incubation
with CHX on neuronal survival was analyzed. Continuous application of CHX
promoted survival for 2-3 days, but thereafter neurons died regardless of
whether CIPE was present or absent. Application of CHX for 6 h from the
onset of the culture was enough to delay the commitment point up to 24 h
after plating, and the addition of CIPE at this time maintained survival
and promoted differentiation of CHX treated neurons. 5. The RNA
transcription blockers Act-D, alpha-amanitin, and DRB were applied to both
trophically deprived and trophically supported neurons, and the survival of
each was evaluated. Neither drug was effective in supporting the survival
of trophically deprived neurons in culture, and in most cases neurons even
when cultured with CIPE died within 1-2 days in the presence of either
drug. 6. Experiments using both CHX and mRNA synthesis blockers were
performed to determine the effect of blocking mRNA transcription in
trophically deprived neurons rescued by CHX. The addition of mRNA synthesis
inhibitors precluded the effect of CHX on neuronal survival. 7. The effect
of CHX (20 micrograms/ml) on RNA and protein synthesis was studied by
measuring the incorporation of radiolabeled metabolic precursors
(3H-leucine or 3H-uridine) into macromolecules. A 95% reduction in the
protein synthesis was observed after 1 h of application of the drug, and by
24 h, 3H-leucine incorporation was reduced to 15-20% of the control values.
Wash out of CHX after 6 h of incubation produced a recovery of protein
synthesis up to 50% of control values 18 h later. CHX did not affect the
synthesis of RNA for up to 12 h; however, it impaired the ability of the
cell to take up metabolic precursors. 8. In conclusion, the present results
support the hypothesis that the CHX effect on neuronal survival is due to
its ability to induce the expression of survival or protective genes rather
than to block the expression of killer proteins. This view is supported by
1) the 24-h delay of the commitment point following the short-term
application of CHX, 2) the impaired ability of CHX to rescue
trophic-deprived neurons by the addition of mRNA synthesis blockers, and 3)
the fact that neuronal survival caused by trophic factors like CIPE, is
blocked by blocking RNA transcription.</description><identifier>ISSN: 0022-3077</identifier><identifier>EISSN: 1522-1598</identifier><identifier>DOI: 10.1152/jn.1995.74.6.2487</identifier><identifier>PMID: 8747208</identifier><language>eng</language><publisher>United States: Am Phys Soc</publisher><subject>Animals ; Cell Death - drug effects ; Cells, Cultured ; Chick Embryo ; Culture Media ; Cycloheximide - pharmacology ; Ganglia, Parasympathetic - cytology ; Ganglia, Parasympathetic - drug effects ; Immunohistochemistry ; Nerve Tissue Proteins - biosynthesis ; Neurons - drug effects ; Protein Synthesis Inhibitors - pharmacology ; RNA, Messenger - biosynthesis ; Transcription, Genetic - drug effects</subject><ispartof>Journal of neurophysiology, 1995-12, Vol.74 (6), p.2487-2499</ispartof><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c359t-26ee785d263e9efbb5a29d67af6c1c404c5ee68af7f92da1811396c1184620ea3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8747208$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bruses, J. L</creatorcontrib><creatorcontrib>Pilar, G. R</creatorcontrib><title>Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture</title><title>Journal of neurophysiology</title><addtitle>J Neurophysiol</addtitle><description>J. L. Bruses and G. R. Pilar
Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA.
1. The relationship between cycloheximide (CHX) and RNA synthesis
inhibitors on trophic-deprived neuronal survival was studied with the use
of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons,
to analyze the biological process of neuronal death caused by trophic
factor withdrawal. Tissue culture conditions were refined by characterizing
the additional medium components required to obtain 100% survival, for at
least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris,
and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly
enriched neuronal cultures almost devoid of nonneuronal cells were used. 2.
The time at which trophically deprived neurons cannot be rescued by the
addition of trophic support, "commitment point," was established to be
between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of
protein translation, reduced 3H-leucine incorporation by 90-95%, at a
concentration of 10-100 micrograms/ml. The effect of the RNA transcription
blockers actinomycin D (Act-D), alpha-amanitin, and 5.6
dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into
macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25%
by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the
3H-uridine incorporation. 4. The effect of short- and long-term incubation
with CHX on neuronal survival was analyzed. Continuous application of CHX
promoted survival for 2-3 days, but thereafter neurons died regardless of
whether CIPE was present or absent. Application of CHX for 6 h from the
onset of the culture was enough to delay the commitment point up to 24 h
after plating, and the addition of CIPE at this time maintained survival
and promoted differentiation of CHX treated neurons. 5. The RNA
transcription blockers Act-D, alpha-amanitin, and DRB were applied to both
trophically deprived and trophically supported neurons, and the survival of
each was evaluated. Neither drug was effective in supporting the survival
of trophically deprived neurons in culture, and in most cases neurons even
when cultured with CIPE died within 1-2 days in the presence of either
drug. 6. Experiments using both CHX and mRNA synthesis blockers were
performed to determine the effect of blocking mRNA transcription in
trophically deprived neurons rescued by CHX. The addition of mRNA synthesis
inhibitors precluded the effect of CHX on neuronal survival. 7. The effect
of CHX (20 micrograms/ml) on RNA and protein synthesis was studied by
measuring the incorporation of radiolabeled metabolic precursors
(3H-leucine or 3H-uridine) into macromolecules. A 95% reduction in the
protein synthesis was observed after 1 h of application of the drug, and by
24 h, 3H-leucine incorporation was reduced to 15-20% of the control values.
Wash out of CHX after 6 h of incubation produced a recovery of protein
synthesis up to 50% of control values 18 h later. CHX did not affect the
synthesis of RNA for up to 12 h; however, it impaired the ability of the
cell to take up metabolic precursors. 8. In conclusion, the present results
support the hypothesis that the CHX effect on neuronal survival is due to
its ability to induce the expression of survival or protective genes rather
than to block the expression of killer proteins. This view is supported by
1) the 24-h delay of the commitment point following the short-term
application of CHX, 2) the impaired ability of CHX to rescue
trophic-deprived neurons by the addition of mRNA synthesis blockers, and 3)
the fact that neuronal survival caused by trophic factors like CIPE, is
blocked by blocking RNA transcription.</description><subject>Animals</subject><subject>Cell Death - drug effects</subject><subject>Cells, Cultured</subject><subject>Chick Embryo</subject><subject>Culture Media</subject><subject>Cycloheximide - pharmacology</subject><subject>Ganglia, Parasympathetic - cytology</subject><subject>Ganglia, Parasympathetic - drug effects</subject><subject>Immunohistochemistry</subject><subject>Nerve Tissue Proteins - biosynthesis</subject><subject>Neurons - drug effects</subject><subject>Protein Synthesis Inhibitors - pharmacology</subject><subject>RNA, Messenger - biosynthesis</subject><subject>Transcription, Genetic - drug effects</subject><issn>0022-3077</issn><issn>1522-1598</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkVFrFDEUhYNY6rb6A3wQ8qRPOyaZySTzWErVQrFQ9DlkMzc7WTLJmsyo019vhl30UQgk3HPOBzcHobeUVJRy9vEQKtp1vBJN1VaskeIF2pQ521LeyZdoQ0h510SIV-gq5wMhRHDCLtGlFI1gRG7Q8521YCYcLTaL8XGA3250PWAdejw-fb3BeQnTANll7MLgdm5yMeByetDTsOamFI-DM9r7pQyPyf2EHhvnnU4L3uuw92siwJxiWCHYzH6aE7xGF1b7DG_O9zX6_unu2-2X7cPj5_vbm4etqXk3bVkLICTvWVtDB3a345p1fSu0bQ01DWkMB2iltsJ2rNdUUlp3RaKyaRkBXV-j9yfuMcUfM-RJjS4b8F4HiHNWQkgpOZX_NVJByr82q5GejCbFnBNYVbYey7qKErUWow5BrcUo0ahWrcWUzLszfN6N0P9NnJso-oeTPrj98MslUMdhyS76uF9W3D_SHxhYmbw</recordid><startdate>19951201</startdate><enddate>19951201</enddate><creator>Bruses, J. L</creator><creator>Pilar, G. R</creator><general>Am Phys Soc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TK</scope><scope>7X8</scope></search><sort><creationdate>19951201</creationdate><title>Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture</title><author>Bruses, J. L ; Pilar, G. R</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c359t-26ee785d263e9efbb5a29d67af6c1c404c5ee68af7f92da1811396c1184620ea3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>Animals</topic><topic>Cell Death - drug effects</topic><topic>Cells, Cultured</topic><topic>Chick Embryo</topic><topic>Culture Media</topic><topic>Cycloheximide - pharmacology</topic><topic>Ganglia, Parasympathetic - cytology</topic><topic>Ganglia, Parasympathetic - drug effects</topic><topic>Immunohistochemistry</topic><topic>Nerve Tissue Proteins - biosynthesis</topic><topic>Neurons - drug effects</topic><topic>Protein Synthesis Inhibitors - pharmacology</topic><topic>RNA, Messenger - biosynthesis</topic><topic>Transcription, Genetic - drug effects</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bruses, J. L</creatorcontrib><creatorcontrib>Pilar, G. R</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Neurosciences Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of neurophysiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bruses, J. L</au><au>Pilar, G. R</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture</atitle><jtitle>Journal of neurophysiology</jtitle><addtitle>J Neurophysiol</addtitle><date>1995-12-01</date><risdate>1995</risdate><volume>74</volume><issue>6</issue><spage>2487</spage><epage>2499</epage><pages>2487-2499</pages><issn>0022-3077</issn><eissn>1522-1598</eissn><abstract>J. L. Bruses and G. R. Pilar
Department of Physiology and Neurobiology, University of Connecticut, Storrs 06269, USA.
1. The relationship between cycloheximide (CHX) and RNA synthesis
inhibitors on trophic-deprived neuronal survival was studied with the use
of primary cultures of stage (St) 34 chick ciliary ganglion (CG) neurons,
to analyze the biological process of neuronal death caused by trophic
factor withdrawal. Tissue culture conditions were refined by characterizing
the additional medium components required to obtain 100% survival, for at
least 1 wk, in the presence of an eye extract [choroid, ciliary body, iris,
and pigment epithelium (CIPE)] as a trophic support for the neurons. Highly
enriched neuronal cultures almost devoid of nonneuronal cells were used. 2.
The time at which trophically deprived neurons cannot be rescued by the
addition of trophic support, "commitment point," was established to be
between 11 and 17 h after trophic deprivation. 3. CHX, an inhibitor of
protein translation, reduced 3H-leucine incorporation by 90-95%, at a
concentration of 10-100 micrograms/ml. The effect of the RNA transcription
blockers actinomycin D (Act-D), alpha-amanitin, and 5.6
dichlorobenzimidazole riboside (DRB) on 3H-uridine incorporation into
macromolecules was evaluated. Total RNA synthesis was inhibited by 10-25%
by alpha-amanitin, whereas Act-D and DRB inhibited 80-97.5% of the
3H-uridine incorporation. 4. The effect of short- and long-term incubation
with CHX on neuronal survival was analyzed. Continuous application of CHX
promoted survival for 2-3 days, but thereafter neurons died regardless of
whether CIPE was present or absent. Application of CHX for 6 h from the
onset of the culture was enough to delay the commitment point up to 24 h
after plating, and the addition of CIPE at this time maintained survival
and promoted differentiation of CHX treated neurons. 5. The RNA
transcription blockers Act-D, alpha-amanitin, and DRB were applied to both
trophically deprived and trophically supported neurons, and the survival of
each was evaluated. Neither drug was effective in supporting the survival
of trophically deprived neurons in culture, and in most cases neurons even
when cultured with CIPE died within 1-2 days in the presence of either
drug. 6. Experiments using both CHX and mRNA synthesis blockers were
performed to determine the effect of blocking mRNA transcription in
trophically deprived neurons rescued by CHX. The addition of mRNA synthesis
inhibitors precluded the effect of CHX on neuronal survival. 7. The effect
of CHX (20 micrograms/ml) on RNA and protein synthesis was studied by
measuring the incorporation of radiolabeled metabolic precursors
(3H-leucine or 3H-uridine) into macromolecules. A 95% reduction in the
protein synthesis was observed after 1 h of application of the drug, and by
24 h, 3H-leucine incorporation was reduced to 15-20% of the control values.
Wash out of CHX after 6 h of incubation produced a recovery of protein
synthesis up to 50% of control values 18 h later. CHX did not affect the
synthesis of RNA for up to 12 h; however, it impaired the ability of the
cell to take up metabolic precursors. 8. In conclusion, the present results
support the hypothesis that the CHX effect on neuronal survival is due to
its ability to induce the expression of survival or protective genes rather
than to block the expression of killer proteins. This view is supported by
1) the 24-h delay of the commitment point following the short-term
application of CHX, 2) the impaired ability of CHX to rescue
trophic-deprived neurons by the addition of mRNA synthesis blockers, and 3)
the fact that neuronal survival caused by trophic factors like CIPE, is
blocked by blocking RNA transcription.</abstract><cop>United States</cop><pub>Am Phys Soc</pub><pmid>8747208</pmid><doi>10.1152/jn.1995.74.6.2487</doi><tpages>13</tpages></addata></record> |
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| ispartof | Journal of neurophysiology, 1995-12, Vol.74 (6), p.2487-2499 |
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| language | eng |
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| source | MEDLINE; Alma/SFX Local Collection |
| subjects | Animals Cell Death - drug effects Cells, Cultured Chick Embryo Culture Media Cycloheximide - pharmacology Ganglia, Parasympathetic - cytology Ganglia, Parasympathetic - drug effects Immunohistochemistry Nerve Tissue Proteins - biosynthesis Neurons - drug effects Protein Synthesis Inhibitors - pharmacology RNA, Messenger - biosynthesis Transcription, Genetic - drug effects |
| title | Effect of cycloheximide and mRNA synthesis inhibition on death of trophically deprived ciliary ganglion neurons in culture |
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