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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 in dissociated spinal cord cultures is precluded by TNF-α–neutralizing antibody. (A) Indirect immunofluorescence labeling with Mac-2 antibody (American Type Culture Collection) showing the abundance of activated microglia in dissociated spinal cord cultures. 30 μm. (B) The effects of TNF-α–neutralizing antibody on the percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was compared. Quadruplet cultures from each embryo were prepared and 5 or 10 μg/ml of TNF-α antibody added to two of the cultures, the other two acting as control. The chart and conjoining table show that inclusion of TNF-α antibody within the culture medium significantly reduced the number of TUNEL-positive DRG neurons in Per cultures (P < 0.001*** by two-way analysis of variance). Addition of 10 μg/ml TNF-α antibody to the culture medium enhanced the protective effect to levels similar to results found for WT. There was no significant effect of TNF-α–neutralizing antibody on WT DRG neuronal viability (unpublished data) Bar, 50 μM.
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fig6: Apoptosis of Per DRG neurons in dissociated spinal cord cultures is precluded by TNF-α–neutralizing antibody. (A) Indirect immunofluorescence labeling with Mac-2 antibody (American Type Culture Collection) showing the abundance of activated microglia in dissociated spinal cord cultures. 30 μm. (B) The effects of TNF-α–neutralizing antibody on the percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was compared. Quadruplet cultures from each embryo were prepared and 5 or 10 μg/ml of TNF-α antibody added to two of the cultures, the other two acting as control. The chart and conjoining table show that inclusion of TNF-α antibody within the culture medium significantly reduced the number of TUNEL-positive DRG neurons in Per cultures (P < 0.001*** by two-way analysis of variance). Addition of 10 μg/ml TNF-α antibody to the culture medium enhanced the protective effect to levels similar to results found for WT. There was no significant effect of TNF-α–neutralizing antibody on WT DRG neuronal viability (unpublished data) Bar, 50 μM.

Mentions: As mentioned earlier, in addition to DRG and motor neurons, the dissociated spinal cord cultures contained a number of other cell types, including astrocytes, fibroblasts, and microglia. Microglia in culture adopt an activated phenotype characterized by hypertrophy and development of shorter, stouter processes compared with resting microglia which have smaller perikarya and are highly ramified (Davis et al., 1994; Streit et al., 1999). In addition to these morphologic changes, activated microglia express phenotypic markers reflective of their immunologic function such as major histocompatibiblty complex class I and II antigens (Gehrmann et al., 1995) and Mac-2 (Reichert and Rotshenker, 1996). There was very strong labeling of dissociated spinal cord cultures with antibody to Mac-2, demonstrating the presence of activated microglia (Fig. 6 A). In contrast, there was only minimal labeling of pure DRG neuronal cultures with Mac-2 antibody (unpublished data), most likely due to contaminating hematogenous macrophages. Although activated astrocytes, revealed by intense immunoreactivity with antibody recognizing glial fibrillary acidic protein, were also present in our cultures (unpublished data), the abundance of activated microglia in dissociated spinal cord cultures represented the most clear difference from pure DRG cultures.


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 in dissociated spinal cord cultures is precluded by TNF-α–neutralizing antibody. (A) Indirect immunofluorescence labeling with Mac-2 antibody (American Type Culture Collection) showing the abundance of activated microglia in dissociated spinal cord cultures. 30 μm. (B) The effects of TNF-α–neutralizing antibody on the percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was compared. Quadruplet cultures from each embryo were prepared and 5 or 10 μg/ml of TNF-α antibody added to two of the cultures, the other two acting as control. The chart and conjoining table show that inclusion of TNF-α antibody within the culture medium significantly reduced the number of TUNEL-positive DRG neurons in Per cultures (P < 0.001*** by two-way analysis of variance). Addition of 10 μg/ml TNF-α antibody to the culture medium enhanced the protective effect to levels similar to results found for WT. There was no significant effect of TNF-α–neutralizing antibody on WT DRG neuronal viability (unpublished data) Bar, 50 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198840&req=5

fig6: Apoptosis of Per DRG neurons in dissociated spinal cord cultures is precluded by TNF-α–neutralizing antibody. (A) Indirect immunofluorescence labeling with Mac-2 antibody (American Type Culture Collection) showing the abundance of activated microglia in dissociated spinal cord cultures. 30 μm. (B) The effects of TNF-α–neutralizing antibody on the percentage of TUNEL-positive DRG neurons in WT and Per dissociated spinal cord cultures was compared. Quadruplet cultures from each embryo were prepared and 5 or 10 μg/ml of TNF-α antibody added to two of the cultures, the other two acting as control. The chart and conjoining table show that inclusion of TNF-α antibody within the culture medium significantly reduced the number of TUNEL-positive DRG neurons in Per cultures (P < 0.001*** by two-way analysis of variance). Addition of 10 μg/ml TNF-α antibody to the culture medium enhanced the protective effect to levels similar to results found for WT. There was no significant effect of TNF-α–neutralizing antibody on WT DRG neuronal viability (unpublished data) Bar, 50 μM.
Mentions: As mentioned earlier, in addition to DRG and motor neurons, the dissociated spinal cord cultures contained a number of other cell types, including astrocytes, fibroblasts, and microglia. Microglia in culture adopt an activated phenotype characterized by hypertrophy and development of shorter, stouter processes compared with resting microglia which have smaller perikarya and are highly ramified (Davis et al., 1994; Streit et al., 1999). In addition to these morphologic changes, activated microglia express phenotypic markers reflective of their immunologic function such as major histocompatibiblty complex class I and II antigens (Gehrmann et al., 1995) and Mac-2 (Reichert and Rotshenker, 1996). There was very strong labeling of dissociated spinal cord cultures with antibody to Mac-2, demonstrating the presence of activated microglia (Fig. 6 A). In contrast, there was only minimal labeling of pure DRG neuronal cultures with Mac-2 antibody (unpublished data), most likely due to contaminating hematogenous macrophages. Although activated astrocytes, revealed by intense immunoreactivity with antibody recognizing glial fibrillary acidic protein, were also present in our cultures (unpublished data), the abundance of activated microglia in dissociated spinal cord cultures represented the most clear difference from pure DRG cultures.

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