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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

Inhibition of ROCK restores neurotransmitter-associated functions in prion-infected 1C115-HT neuronal cells.(A) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in Fk-infected 1C11 cells treated or not with dimethylfasudil (2 μM) or Y-27632 (100 μM) for 1h versus uninfected 1C11 cells. (B) 5-HT synthesis (TPH activity) and TPH immunoblotting, (C) transport by SERT (paroxetine binding) and SERT immunoblotting, (D) storage (VMAT-2 tetrabenazine binding), (E) intracellular content, and (F) catabolism (5-HIAA concentration) in uninfected 1C115-HT cells and Fk-infected 1C115-HT cells differentiated for 4 days along the serotonergic pathway in the absence or presence of Y-27632 (100 μM) or dimethylfasudil (2 μM). (G) Concentration of 5-HT-derived oxidized species in cell lysates of 1C115-HT cells and Fk-1C115-HT differentiated in the absence or presence of Y-27632 or dimethylfasudil. n = 6 for each condition. Values are the mean ± s.e.m. # P < 0.05 versus uninfected cells. ## P < 0.05 versus infected cells.
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ppat.1005073.g002: Inhibition of ROCK restores neurotransmitter-associated functions in prion-infected 1C115-HT neuronal cells.(A) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in Fk-infected 1C11 cells treated or not with dimethylfasudil (2 μM) or Y-27632 (100 μM) for 1h versus uninfected 1C11 cells. (B) 5-HT synthesis (TPH activity) and TPH immunoblotting, (C) transport by SERT (paroxetine binding) and SERT immunoblotting, (D) storage (VMAT-2 tetrabenazine binding), (E) intracellular content, and (F) catabolism (5-HIAA concentration) in uninfected 1C115-HT cells and Fk-infected 1C115-HT cells differentiated for 4 days along the serotonergic pathway in the absence or presence of Y-27632 (100 μM) or dimethylfasudil (2 μM). (G) Concentration of 5-HT-derived oxidized species in cell lysates of 1C115-HT cells and Fk-1C115-HT differentiated in the absence or presence of Y-27632 or dimethylfasudil. n = 6 for each condition. Values are the mean ± s.e.m. # P < 0.05 versus uninfected cells. ## P < 0.05 versus infected cells.

Mentions: We next wondered whether inhibiting ROCK activity would counteract prion-induced neuritogenesis defects. When treated with two distinct ROCK inhibitors, namely Y-27632 (100 μM) or dimethylfasudil (2 μM) (for review see [30–32] and references therein), ~90% of Fk- or 22L-infected 1C11 cells induced to differentiate along the serotonergic pathway developed neurites. At the end of the program (day 4), prion-infected 1C11 cells differentiated in the presence of ROCK inhibitors (referred to as Rocki-infected 1C115-HT cells) exhibited neurites that were thin, bipolar and extended from the ovoid cell body as for uninfected 1C115-HT neuronal cells (S1 Fig and S1 Table). The mean neurite length of Rocki-infected 1C115-HT cells was comparable to that of uninfected 1C115-HT cells differentiated in the presence of ROCK inhibitors (S1 Table). Rescued neuritogenesis by Fk-infected 1C11 precursor cells upon ROCK inhibition was associated with decrease to basal level of phospho-cofilin level on Ser 3 (Fig 2A), which restores cofilin severing activity towards F-actin and renders Fk-1C11 precursor cells competent to sprout neurites when induced towards the neuronal program [15].


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)

Inhibition of ROCK restores neurotransmitter-associated functions in prion-infected 1C115-HT neuronal cells.(A) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in Fk-infected 1C11 cells treated or not with dimethylfasudil (2 μM) or Y-27632 (100 μM) for 1h versus uninfected 1C11 cells. (B) 5-HT synthesis (TPH activity) and TPH immunoblotting, (C) transport by SERT (paroxetine binding) and SERT immunoblotting, (D) storage (VMAT-2 tetrabenazine binding), (E) intracellular content, and (F) catabolism (5-HIAA concentration) in uninfected 1C115-HT cells and Fk-infected 1C115-HT cells differentiated for 4 days along the serotonergic pathway in the absence or presence of Y-27632 (100 μM) or dimethylfasudil (2 μM). (G) Concentration of 5-HT-derived oxidized species in cell lysates of 1C115-HT cells and Fk-1C115-HT differentiated in the absence or presence of Y-27632 or dimethylfasudil. n = 6 for each condition. Values are the mean ± s.e.m. # P < 0.05 versus uninfected cells. ## P < 0.05 versus infected cells.
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Related In: Results  -  Collection

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ppat.1005073.g002: Inhibition of ROCK restores neurotransmitter-associated functions in prion-infected 1C115-HT neuronal cells.(A) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in Fk-infected 1C11 cells treated or not with dimethylfasudil (2 μM) or Y-27632 (100 μM) for 1h versus uninfected 1C11 cells. (B) 5-HT synthesis (TPH activity) and TPH immunoblotting, (C) transport by SERT (paroxetine binding) and SERT immunoblotting, (D) storage (VMAT-2 tetrabenazine binding), (E) intracellular content, and (F) catabolism (5-HIAA concentration) in uninfected 1C115-HT cells and Fk-infected 1C115-HT cells differentiated for 4 days along the serotonergic pathway in the absence or presence of Y-27632 (100 μM) or dimethylfasudil (2 μM). (G) Concentration of 5-HT-derived oxidized species in cell lysates of 1C115-HT cells and Fk-1C115-HT differentiated in the absence or presence of Y-27632 or dimethylfasudil. n = 6 for each condition. Values are the mean ± s.e.m. # P < 0.05 versus uninfected cells. ## P < 0.05 versus infected cells.
Mentions: We next wondered whether inhibiting ROCK activity would counteract prion-induced neuritogenesis defects. When treated with two distinct ROCK inhibitors, namely Y-27632 (100 μM) or dimethylfasudil (2 μM) (for review see [30–32] and references therein), ~90% of Fk- or 22L-infected 1C11 cells induced to differentiate along the serotonergic pathway developed neurites. At the end of the program (day 4), prion-infected 1C11 cells differentiated in the presence of ROCK inhibitors (referred to as Rocki-infected 1C115-HT cells) exhibited neurites that were thin, bipolar and extended from the ovoid cell body as for uninfected 1C115-HT neuronal cells (S1 Fig and S1 Table). The mean neurite length of Rocki-infected 1C115-HT cells was comparable to that of uninfected 1C115-HT cells differentiated in the presence of ROCK inhibitors (S1 Table). Rescued neuritogenesis by Fk-infected 1C11 precursor cells upon ROCK inhibition was associated with decrease to basal level of phospho-cofilin level on Ser 3 (Fig 2A), which restores cofilin severing activity towards F-actin and renders Fk-1C11 precursor cells competent to sprout neurites when induced towards the neuronal program [15].

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