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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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Overexpression of peripherin induces apoptosis of DRG neurons. (A) TUNEL assays were performed on WT and Per dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with peripherin antibody so that TUNEL-positive DRG neurons could be correlated with the presence of peripherin aggregates. TUNEL-positive DRG neurons were shrunken and had condensed chromatin and an intact plasma membrane, consistent with the changes associated with apoptosis. (B) Dissociated spinal cord cultures were labeled by indirect immunofluorescence with antibody recognizing activated caspase-3 (C-3). A number of DRG neurons in Per cultures were labeled with C-3. No labeling of WT DRG neurons was observed. (C) Electron microscopic evaluation of apoptotic Per DRG neurons showed that there was severe disruption of mitochondrial cristae (arrowheads). (D) Comparison of specific apoptosis of Per DRG neurons in dissociated spinal cord cultures from three separate litters. The percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was calculated after 14 d in vitro. Specific apoptosis was calculated as the percentage of TUNEL-positive Per DRGs, minus the percentage of TUNEL-positive WT DRGs/100, minus the percentage of TUNEL-positive WT DRGs. Specific apoptosis of Per DRG neurons was usually between 30 and 40%. The results shown are from three litters and are typical of our findings. Bars: (A) 20 μM; (B) 15 μM; (C) 0.4 μM.
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fig4: Overexpression of peripherin induces apoptosis of DRG neurons. (A) TUNEL assays were performed on WT and Per dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with peripherin antibody so that TUNEL-positive DRG neurons could be correlated with the presence of peripherin aggregates. TUNEL-positive DRG neurons were shrunken and had condensed chromatin and an intact plasma membrane, consistent with the changes associated with apoptosis. (B) Dissociated spinal cord cultures were labeled by indirect immunofluorescence with antibody recognizing activated caspase-3 (C-3). A number of DRG neurons in Per cultures were labeled with C-3. No labeling of WT DRG neurons was observed. (C) Electron microscopic evaluation of apoptotic Per DRG neurons showed that there was severe disruption of mitochondrial cristae (arrowheads). (D) Comparison of specific apoptosis of Per DRG neurons in dissociated spinal cord cultures from three separate litters. The percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was calculated after 14 d in vitro. Specific apoptosis was calculated as the percentage of TUNEL-positive Per DRGs, minus the percentage of TUNEL-positive WT DRGs/100, minus the percentage of TUNEL-positive WT DRGs. Specific apoptosis of Per DRG neurons was usually between 30 and 40%. The results shown are from three litters and are typical of our findings. Bars: (A) 20 μM; (B) 15 μM; (C) 0.4 μM.

Mentions: By phase contrast microscopy, DRG neurons in Per cultures appeared shrunken and became detached from the substratum, characteristic features of apoptosis (Majno and Joris, 1995). To clarify if apoptosis was indeed occurring, we performed TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays on Per cultures and compared the number of TUNEL-positive DRG neurons with those found in WT cultures. We found that there was a dramatic increase in TUNEL-positive DRG neurons in Per cultures. The percentage specific apoptosis of Per DRG neurons was calculated as 32.8% ± 3.5% (calculated from counts obtained from three separate litters; Fig. 4 D), and in general was found to range between 30 and 40%. After TUNEL labeling, cultures were labeled for indirect immunofluorescence with antibody to peripherin to determine if there was a correlation between the presence of peripherin aggregates and apoptosis (Fig. 4 A). Indeed, it was found that all DRG neurons in Per cultures contained peripherin aggregates.


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)

Overexpression of peripherin induces apoptosis of DRG neurons. (A) TUNEL assays were performed on WT and Per dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with peripherin antibody so that TUNEL-positive DRG neurons could be correlated with the presence of peripherin aggregates. TUNEL-positive DRG neurons were shrunken and had condensed chromatin and an intact plasma membrane, consistent with the changes associated with apoptosis. (B) Dissociated spinal cord cultures were labeled by indirect immunofluorescence with antibody recognizing activated caspase-3 (C-3). A number of DRG neurons in Per cultures were labeled with C-3. No labeling of WT DRG neurons was observed. (C) Electron microscopic evaluation of apoptotic Per DRG neurons showed that there was severe disruption of mitochondrial cristae (arrowheads). (D) Comparison of specific apoptosis of Per DRG neurons in dissociated spinal cord cultures from three separate litters. The percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was calculated after 14 d in vitro. Specific apoptosis was calculated as the percentage of TUNEL-positive Per DRGs, minus the percentage of TUNEL-positive WT DRGs/100, minus the percentage of TUNEL-positive WT DRGs. Specific apoptosis of Per DRG neurons was usually between 30 and 40%. The results shown are from three litters and are typical of our findings. Bars: (A) 20 μM; (B) 15 μM; (C) 0.4 μM.
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Related In: Results  -  Collection

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fig4: Overexpression of peripherin induces apoptosis of DRG neurons. (A) TUNEL assays were performed on WT and Per dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with peripherin antibody so that TUNEL-positive DRG neurons could be correlated with the presence of peripherin aggregates. TUNEL-positive DRG neurons were shrunken and had condensed chromatin and an intact plasma membrane, consistent with the changes associated with apoptosis. (B) Dissociated spinal cord cultures were labeled by indirect immunofluorescence with antibody recognizing activated caspase-3 (C-3). A number of DRG neurons in Per cultures were labeled with C-3. No labeling of WT DRG neurons was observed. (C) Electron microscopic evaluation of apoptotic Per DRG neurons showed that there was severe disruption of mitochondrial cristae (arrowheads). (D) Comparison of specific apoptosis of Per DRG neurons in dissociated spinal cord cultures from three separate litters. The percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was calculated after 14 d in vitro. Specific apoptosis was calculated as the percentage of TUNEL-positive Per DRGs, minus the percentage of TUNEL-positive WT DRGs/100, minus the percentage of TUNEL-positive WT DRGs. Specific apoptosis of Per DRG neurons was usually between 30 and 40%. The results shown are from three litters and are typical of our findings. Bars: (A) 20 μM; (B) 15 μM; (C) 0.4 μM.
Mentions: By phase contrast microscopy, DRG neurons in Per cultures appeared shrunken and became detached from the substratum, characteristic features of apoptosis (Majno and Joris, 1995). To clarify if apoptosis was indeed occurring, we performed TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays on Per cultures and compared the number of TUNEL-positive DRG neurons with those found in WT cultures. We found that there was a dramatic increase in TUNEL-positive DRG neurons in Per cultures. The percentage specific apoptosis of Per DRG neurons was calculated as 32.8% ± 3.5% (calculated from counts obtained from three separate litters; Fig. 4 D), and in general was found to range between 30 and 40%. After TUNEL labeling, cultures were labeled for indirect immunofluorescence with antibody to peripherin to determine if there was a correlation between the presence of peripherin aggregates and apoptosis (Fig. 4 A). Indeed, it was found that all DRG neurons in Per cultures contained peripherin aggregates.

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