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Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation.

Alleaume-Butaux A, Nicot S, Pietri M, Baudry A, Dakowski C, Tixador P, Ardila-Osorio H, Haeberlé AM, Bailly Y, Peyrin JM, Launay JM, Kellermann O, Schneider B - PLoS Pathog. (2015)

Bottom Line: This overactivation of ROCK also disturbed overall neurotransmitter-associated functions.In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc.In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR-S 1124, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1124, Paris, France.

ABSTRACT
In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.

No MeSH data available.


Related in: MedlinePlus

ROCK-I interacts with PDK1 and phosphorylates PDK1.(A) Immunoprecipitation of ROCK-I and immunoblotting of PDK1 in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. (B) Cell 32P metabolic labeling followed by PDK1 immunoprecipitation and western blotting for PDK1 phosphorylation level in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. Values are the mean ± s.e.m. # P < 0.05 versus non treated uninfected cells. ## P < 0.05 versus non treated infected cells.
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ppat.1005073.g005: ROCK-I interacts with PDK1 and phosphorylates PDK1.(A) Immunoprecipitation of ROCK-I and immunoblotting of PDK1 in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. (B) Cell 32P metabolic labeling followed by PDK1 immunoprecipitation and western blotting for PDK1 phosphorylation level in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. Values are the mean ± s.e.m. # P < 0.05 versus non treated uninfected cells. ## P < 0.05 versus non treated infected cells.

Mentions: The acute regulation of PDK1 activity relies on a complex interplay between PDK1 phosphorylation, subcellular localization, binding of regulators and conformational changes (for review see [41] and references therein). To investigate how ROCK-I regulates PDK1 activity, we designed a first set of experiments centered on the interaction between ROCK-I and PDK1. In uninfected 1C115-HT cells, ROCK-I immunoprecipitation followed by PDK1 western blotting revealed that ROCK-I interacts with PDK1 (Fig 5A). In Fk-infected 1C115-HT cells, the fraction of PDK1 interacting with ROCK-I was ~2-fold increased compared to uninfected cells (Fig 5A). Of note, the level of ROCK-I did not vary between uninfected 1C115-HT and Fk-1C115-HT cells (Fig 5A). The rise in ROCK activity induced by PrPSc thus increases the number of PDK1 molecules recruited by ROCK-I. Exposure of uninfected 1C115-HT or Fk-1C115-HT cells to Y-27632 (100 μM) for 1h dissociated the ROCK-I / PDK1 complex by ~70% and ~90%, respectively (Fig 5A), further indicating that the ROCK-I-kinase activity is necessary for its association with PDK1.


Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation.

Alleaume-Butaux A, Nicot S, Pietri M, Baudry A, Dakowski C, Tixador P, Ardila-Osorio H, Haeberlé AM, Bailly Y, Peyrin JM, Launay JM, Kellermann O, Schneider B - PLoS Pathog. (2015)

ROCK-I interacts with PDK1 and phosphorylates PDK1.(A) Immunoprecipitation of ROCK-I and immunoblotting of PDK1 in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. (B) Cell 32P metabolic labeling followed by PDK1 immunoprecipitation and western blotting for PDK1 phosphorylation level in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. Values are the mean ± s.e.m. # P < 0.05 versus non treated uninfected cells. ## P < 0.05 versus non treated infected cells.
© Copyright Policy
Related In: Results  -  Collection

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ppat.1005073.g005: ROCK-I interacts with PDK1 and phosphorylates PDK1.(A) Immunoprecipitation of ROCK-I and immunoblotting of PDK1 in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. (B) Cell 32P metabolic labeling followed by PDK1 immunoprecipitation and western blotting for PDK1 phosphorylation level in uninfected 1C115-HT or Fk-1C115-HT treated or not with Y-27632 (100 μM, 1h) as well as in 1C115-HT transfected with S241A PDK1 mutant. Values are the mean ± s.e.m. # P < 0.05 versus non treated uninfected cells. ## P < 0.05 versus non treated infected cells.
Mentions: The acute regulation of PDK1 activity relies on a complex interplay between PDK1 phosphorylation, subcellular localization, binding of regulators and conformational changes (for review see [41] and references therein). To investigate how ROCK-I regulates PDK1 activity, we designed a first set of experiments centered on the interaction between ROCK-I and PDK1. In uninfected 1C115-HT cells, ROCK-I immunoprecipitation followed by PDK1 western blotting revealed that ROCK-I interacts with PDK1 (Fig 5A). In Fk-infected 1C115-HT cells, the fraction of PDK1 interacting with ROCK-I was ~2-fold increased compared to uninfected cells (Fig 5A). Of note, the level of ROCK-I did not vary between uninfected 1C115-HT and Fk-1C115-HT cells (Fig 5A). The rise in ROCK activity induced by PrPSc thus increases the number of PDK1 molecules recruited by ROCK-I. Exposure of uninfected 1C115-HT or Fk-1C115-HT cells to Y-27632 (100 μM) for 1h dissociated the ROCK-I / PDK1 complex by ~70% and ~90%, respectively (Fig 5A), further indicating that the ROCK-I-kinase activity is necessary for its association with PDK1.

Bottom Line: This overactivation of ROCK also disturbed overall neurotransmitter-associated functions.In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc.In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR-S 1124, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1124, Paris, France.

ABSTRACT
In prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases.

No MeSH data available.


Related in: MedlinePlus