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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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Intranuclear microinjection of the peripherin gene induces death of cultured motor neurons. (A) Mammalian expression plasmids encoding either NF-L or peripherin were microinjected into the nuclei of motor neurons in dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with antibody recognizing NF-L (NR4) or peripherin (MAB1527). The results in this figure show the organization of NF-L or peripherin in microinjected motor neurons after 3 d of expression. Overexpression of NF-L led to intense immunofluorescence labeling of motor neurons, characterized by the formation of coiling loops in the perikaryon. In contrast, overexpressed peripherin did not integrate normally into the existing cytoplasmic network, but instead formed punctate aggregates that were also clearly apparent in neurites. (B) Double immunofluorescence labeling with antibodies to peripherin (MAB1527) and NF-L (AB1983) of the neurites from a motor neuron that had been microinjected with peripherin, showing the disruption, by peripherin, of the endogenous neurofilament network (arrows). (C) Motor neurons in dissociated spinal cord cultures were microinjected with expression plasmids encoding (a) peripherin; (b) NF-L; (c) NF-L and peripherin; or (d) expression plasmid alone (pRcCMV; control). The number of viable motor neurons was counted each day for 7 d, and the results from each microinjection experiment were compared. The chart shows that peripherin was extremely toxic to motor neurons. In contrast, there was no significant effect of NF-L overexpression on viability (Student's t test; P < 0.05). Indeed, comicroinjection of NF-L with peripherin offered protection from the toxic effects observed with peripherin microinjection alone. Bars: (A) 25 μm; (B) 15 μm.
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fig1: Intranuclear microinjection of the peripherin gene induces death of cultured motor neurons. (A) Mammalian expression plasmids encoding either NF-L or peripherin were microinjected into the nuclei of motor neurons in dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with antibody recognizing NF-L (NR4) or peripherin (MAB1527). The results in this figure show the organization of NF-L or peripherin in microinjected motor neurons after 3 d of expression. Overexpression of NF-L led to intense immunofluorescence labeling of motor neurons, characterized by the formation of coiling loops in the perikaryon. In contrast, overexpressed peripherin did not integrate normally into the existing cytoplasmic network, but instead formed punctate aggregates that were also clearly apparent in neurites. (B) Double immunofluorescence labeling with antibodies to peripherin (MAB1527) and NF-L (AB1983) of the neurites from a motor neuron that had been microinjected with peripherin, showing the disruption, by peripherin, of the endogenous neurofilament network (arrows). (C) Motor neurons in dissociated spinal cord cultures were microinjected with expression plasmids encoding (a) peripherin; (b) NF-L; (c) NF-L and peripherin; or (d) expression plasmid alone (pRcCMV; control). The number of viable motor neurons was counted each day for 7 d, and the results from each microinjection experiment were compared. The chart shows that peripherin was extremely toxic to motor neurons. In contrast, there was no significant effect of NF-L overexpression on viability (Student's t test; P < 0.05). Indeed, comicroinjection of NF-L with peripherin offered protection from the toxic effects observed with peripherin microinjection alone. Bars: (A) 25 μm; (B) 15 μm.

Mentions: In control cultures, either nonmicroinjected or microinjected with vector alone, there was minimal labeling of motor neurons with peripherin antibody (unpublished data). However, in motor neurons microinjected with the peripherin expression plasmid, there was intense peripherin immunoreactivity (Fig. 1 A). Interestingly, peripherin did not integrate normally into the existing cytoplasmic intermediate filament network, but instead formed clearly defined punctate aggregates that filled the cell body and were also present in neurites (Fig. 1 A). In contrast, the plasmid-derived NF-L protein appeared to integrate into the existing network, forming looping coils of filaments after 3 d that progressed to substantial perikaryal aggregates after 7 d of expression (Fig. 1 A). Motor neurons microinjected with the peripherin expression plasmid were double labeled with peripherin and neurofilament antibodies (Fig. 1 B; similar findings obtained with antibody to neurofilament medium subunit [NF-M]), showing that peripherin caused disruption of the existing neurofilament network.


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)

Intranuclear microinjection of the peripherin gene induces death of cultured motor neurons. (A) Mammalian expression plasmids encoding either NF-L or peripherin were microinjected into the nuclei of motor neurons in dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with antibody recognizing NF-L (NR4) or peripherin (MAB1527). The results in this figure show the organization of NF-L or peripherin in microinjected motor neurons after 3 d of expression. Overexpression of NF-L led to intense immunofluorescence labeling of motor neurons, characterized by the formation of coiling loops in the perikaryon. In contrast, overexpressed peripherin did not integrate normally into the existing cytoplasmic network, but instead formed punctate aggregates that were also clearly apparent in neurites. (B) Double immunofluorescence labeling with antibodies to peripherin (MAB1527) and NF-L (AB1983) of the neurites from a motor neuron that had been microinjected with peripherin, showing the disruption, by peripherin, of the endogenous neurofilament network (arrows). (C) Motor neurons in dissociated spinal cord cultures were microinjected with expression plasmids encoding (a) peripherin; (b) NF-L; (c) NF-L and peripherin; or (d) expression plasmid alone (pRcCMV; control). The number of viable motor neurons was counted each day for 7 d, and the results from each microinjection experiment were compared. The chart shows that peripherin was extremely toxic to motor neurons. In contrast, there was no significant effect of NF-L overexpression on viability (Student's t test; P < 0.05). Indeed, comicroinjection of NF-L with peripherin offered protection from the toxic effects observed with peripherin microinjection alone. Bars: (A) 25 μm; (B) 15 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198840&req=5

fig1: Intranuclear microinjection of the peripherin gene induces death of cultured motor neurons. (A) Mammalian expression plasmids encoding either NF-L or peripherin were microinjected into the nuclei of motor neurons in dissociated spinal cord cultures. Cultures were then labeled by indirect immunofluorescence with antibody recognizing NF-L (NR4) or peripherin (MAB1527). The results in this figure show the organization of NF-L or peripherin in microinjected motor neurons after 3 d of expression. Overexpression of NF-L led to intense immunofluorescence labeling of motor neurons, characterized by the formation of coiling loops in the perikaryon. In contrast, overexpressed peripherin did not integrate normally into the existing cytoplasmic network, but instead formed punctate aggregates that were also clearly apparent in neurites. (B) Double immunofluorescence labeling with antibodies to peripherin (MAB1527) and NF-L (AB1983) of the neurites from a motor neuron that had been microinjected with peripherin, showing the disruption, by peripherin, of the endogenous neurofilament network (arrows). (C) Motor neurons in dissociated spinal cord cultures were microinjected with expression plasmids encoding (a) peripherin; (b) NF-L; (c) NF-L and peripherin; or (d) expression plasmid alone (pRcCMV; control). The number of viable motor neurons was counted each day for 7 d, and the results from each microinjection experiment were compared. The chart shows that peripherin was extremely toxic to motor neurons. In contrast, there was no significant effect of NF-L overexpression on viability (Student's t test; P < 0.05). Indeed, comicroinjection of NF-L with peripherin offered protection from the toxic effects observed with peripherin microinjection alone. Bars: (A) 25 μm; (B) 15 μm.
Mentions: In control cultures, either nonmicroinjected or microinjected with vector alone, there was minimal labeling of motor neurons with peripherin antibody (unpublished data). However, in motor neurons microinjected with the peripherin expression plasmid, there was intense peripherin immunoreactivity (Fig. 1 A). Interestingly, peripherin did not integrate normally into the existing cytoplasmic intermediate filament network, but instead formed clearly defined punctate aggregates that filled the cell body and were also present in neurites (Fig. 1 A). In contrast, the plasmid-derived NF-L protein appeared to integrate into the existing network, forming looping coils of filaments after 3 d that progressed to substantial perikaryal aggregates after 7 d of expression (Fig. 1 A). Motor neurons microinjected with the peripherin expression plasmid were double labeled with peripherin and neurofilament antibodies (Fig. 1 B; similar findings obtained with antibody to neurofilament medium subunit [NF-M]), showing that peripherin caused disruption of the existing neurofilament network.

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