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Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-alpha.

Robertson J, Beaulieu JM, Doroudchi MM, Durham HD, Julien JP, Mushynski WE - J. Cell Biol. (2001)

Bottom Line: To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures.Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha.This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neurosciences, Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada.

ABSTRACT
Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.

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Apoptosis of Per DRG neurons requires CNS cell culture environment. (A) It was observed that localized groupings of Per DRG neurons within dissociated spinal cord cultures were largely TUNEL negative despite containing peripherin aggregates. Double labeling of such a grouping with TUNEL and with peripherin antibody shows that all the DRG neurons in the field have peripherin aggregates, but that only a few are TUNEL positive and have condensed chromatin (arrows). (B) Pure DRG neuronal cultures were prepared from both WT and Per transgenic embryos, and the number of TUNEL-positive neurons was compared with the findings from dissociated spinal cord cultures (spinal culture). The chart and conjoining table show that there was no significant increase in the number of TUNEL-positive DRG neurons in pure Per DRG cultures compared with WT DRG neurons (DRG culture). To investigate the effect of the cell culture environment on Per DRG viability, pure DRG neurons were grown for 4 d on glass coverslips and then placed in inserts in plates that had (Coculture) or had not (coculture control) been seeded with dissociated spinal cord cultures. Our findings show that when pure cultures of Per DRG neurons were cocultured with dissociated spinal cord cultures, there was a significant increase in apoptosis compared with control P < 0.001*** (by two-way analysis of variance). (C) A comparison of TUNEL labeling of pure cultures of Per DRGs, cocultured in the absence (coculture control) or presence of dissociated spinal cord cells (coculture). The arrows indicate DRG neurons. Note the increase in number of TUNEL-positive DRG neurons and their shrunken appearance in spinal cord coculture compared with control DRG neurons (arrows). Bars: (A) 50 μM; (C) 50 μM.
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fig5: Apoptosis of Per DRG neurons requires CNS cell culture environment. (A) It was observed that localized groupings of Per DRG neurons within dissociated spinal cord cultures were largely TUNEL negative despite containing peripherin aggregates. Double labeling of such a grouping with TUNEL and with peripherin antibody shows that all the DRG neurons in the field have peripherin aggregates, but that only a few are TUNEL positive and have condensed chromatin (arrows). (B) Pure DRG neuronal cultures were prepared from both WT and Per transgenic embryos, and the number of TUNEL-positive neurons was compared with the findings from dissociated spinal cord cultures (spinal culture). The chart and conjoining table show that there was no significant increase in the number of TUNEL-positive DRG neurons in pure Per DRG cultures compared with WT DRG neurons (DRG culture). To investigate the effect of the cell culture environment on Per DRG viability, pure DRG neurons were grown for 4 d on glass coverslips and then placed in inserts in plates that had (Coculture) or had not (coculture control) been seeded with dissociated spinal cord cultures. Our findings show that when pure cultures of Per DRG neurons were cocultured with dissociated spinal cord cultures, there was a significant increase in apoptosis compared with control P < 0.001*** (by two-way analysis of variance). (C) A comparison of TUNEL labeling of pure cultures of Per DRGs, cocultured in the absence (coculture control) or presence of dissociated spinal cord cells (coculture). The arrows indicate DRG neurons. Note the increase in number of TUNEL-positive DRG neurons and their shrunken appearance in spinal cord coculture compared with control DRG neurons (arrows). Bars: (A) 50 μM; (C) 50 μM.

Mentions: In Per-dissociated spinal cord cultures, we observed that in localized groupings of DRG neurons, very few (if any) of the neurons were TUNEL positive, despite the presence of peripherin aggregates (Fig. 5 A). To further investigate this, we compared TUNEL reactivity between pure DRG cultures and mixed spinal cord cultures derived from both WT and Per transgenic embryos. Consistent with our findings in vivo, we found that peripherin aggregates did not induce apoptosis of Per DRG neurons in pure DRG cultures. There was no significant difference between the number of TUNEL-positive DRG neurons in Per or WT DRG cultures (Fig. 5 B). A direct comparison of pure DRG cultures and spinal cultures derived from the same Per embryonic spinal cords (prepared by removing 50% of DRGs and culturing them separately from the remaining spinal cord) demonstrated that apoptosis of Per DRG neurons only occurred in the mixed cell environment of dissociated spinal cord cultures (Fig. 5 B).


Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-alpha.

Robertson J, Beaulieu JM, Doroudchi MM, Durham HD, Julien JP, Mushynski WE - J. Cell Biol. (2001)

Apoptosis of Per DRG neurons requires CNS cell culture environment. (A) It was observed that localized groupings of Per DRG neurons within dissociated spinal cord cultures were largely TUNEL negative despite containing peripherin aggregates. Double labeling of such a grouping with TUNEL and with peripherin antibody shows that all the DRG neurons in the field have peripherin aggregates, but that only a few are TUNEL positive and have condensed chromatin (arrows). (B) Pure DRG neuronal cultures were prepared from both WT and Per transgenic embryos, and the number of TUNEL-positive neurons was compared with the findings from dissociated spinal cord cultures (spinal culture). The chart and conjoining table show that there was no significant increase in the number of TUNEL-positive DRG neurons in pure Per DRG cultures compared with WT DRG neurons (DRG culture). To investigate the effect of the cell culture environment on Per DRG viability, pure DRG neurons were grown for 4 d on glass coverslips and then placed in inserts in plates that had (Coculture) or had not (coculture control) been seeded with dissociated spinal cord cultures. Our findings show that when pure cultures of Per DRG neurons were cocultured with dissociated spinal cord cultures, there was a significant increase in apoptosis compared with control P < 0.001*** (by two-way analysis of variance). (C) A comparison of TUNEL labeling of pure cultures of Per DRGs, cocultured in the absence (coculture control) or presence of dissociated spinal cord cells (coculture). The arrows indicate DRG neurons. Note the increase in number of TUNEL-positive DRG neurons and their shrunken appearance in spinal cord coculture compared with control DRG neurons (arrows). Bars: (A) 50 μM; (C) 50 μM.
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Related In: Results  -  Collection

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fig5: Apoptosis of Per DRG neurons requires CNS cell culture environment. (A) It was observed that localized groupings of Per DRG neurons within dissociated spinal cord cultures were largely TUNEL negative despite containing peripherin aggregates. Double labeling of such a grouping with TUNEL and with peripherin antibody shows that all the DRG neurons in the field have peripherin aggregates, but that only a few are TUNEL positive and have condensed chromatin (arrows). (B) Pure DRG neuronal cultures were prepared from both WT and Per transgenic embryos, and the number of TUNEL-positive neurons was compared with the findings from dissociated spinal cord cultures (spinal culture). The chart and conjoining table show that there was no significant increase in the number of TUNEL-positive DRG neurons in pure Per DRG cultures compared with WT DRG neurons (DRG culture). To investigate the effect of the cell culture environment on Per DRG viability, pure DRG neurons were grown for 4 d on glass coverslips and then placed in inserts in plates that had (Coculture) or had not (coculture control) been seeded with dissociated spinal cord cultures. Our findings show that when pure cultures of Per DRG neurons were cocultured with dissociated spinal cord cultures, there was a significant increase in apoptosis compared with control P < 0.001*** (by two-way analysis of variance). (C) A comparison of TUNEL labeling of pure cultures of Per DRGs, cocultured in the absence (coculture control) or presence of dissociated spinal cord cells (coculture). The arrows indicate DRG neurons. Note the increase in number of TUNEL-positive DRG neurons and their shrunken appearance in spinal cord coculture compared with control DRG neurons (arrows). Bars: (A) 50 μM; (C) 50 μM.
Mentions: In Per-dissociated spinal cord cultures, we observed that in localized groupings of DRG neurons, very few (if any) of the neurons were TUNEL positive, despite the presence of peripherin aggregates (Fig. 5 A). To further investigate this, we compared TUNEL reactivity between pure DRG cultures and mixed spinal cord cultures derived from both WT and Per transgenic embryos. Consistent with our findings in vivo, we found that peripherin aggregates did not induce apoptosis of Per DRG neurons in pure DRG cultures. There was no significant difference between the number of TUNEL-positive DRG neurons in Per or WT DRG cultures (Fig. 5 B). A direct comparison of pure DRG cultures and spinal cultures derived from the same Per embryonic spinal cords (prepared by removing 50% of DRGs and culturing them separately from the remaining spinal cord) demonstrated that apoptosis of Per DRG neurons only occurred in the mixed cell environment of dissociated spinal cord cultures (Fig. 5 B).

Bottom Line: To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures.Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha.This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Neurosciences, Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada.

ABSTRACT
Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.

Show MeSH
Related in: MedlinePlus