Limits...
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 inhibition with Y-27632 attenuates prion disease in mice.(A) Cerebellar immunoperoxidase staining to visualize PrPSc deposition in the cerebellar cortex (CBCX) and deep cerebellar nuclei (DCN) of 22L-infected mice, Scale bars, 100 μm. (B) Immunoperoxidase staining to visualize phosphorylated cofilin on Ser3 in CBCX and DCN of mock-inoculated (SHAM) and 22L-infected mice. Scale bars, 100 μm. (C) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in 22L-infected mice infused or not with Y-27632 versus SHAM mice. n = 4 in triplicate. (D) Survival curves of SHAM and 22L-inoculated mice via the intracerebellar route (i.c.b.) infused or not with the ROCK inhibitor Y-27632 by intraperitoneal injection (i.p.) starting at 130 days after infection (5 mg per kg body weight per day; 0.25 μl h-1). n = 10 mice per group. (E) Static rod test between 130 and 160 days after infection in 22L-infected mice treated with Y-27632. n = 10 mice per group. (F) Left, Western-blot for proteinase K-resistant PrPSc in brain extracts from SHAM and 22L-infected mice infused or not with Y-27632. Right, post-mortem quantification of proteinase K-resistant PrPSc in brains of 22L-infected mice treated or not with Y-27632. n = 10 for each condition. (G) PDK1 activity in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. (H) Immunoblot analysis of sucrose gradient fractions of cerebellar extracts of SHAM mice and 22L-infected mice infused or not with Y-27632 to assess TACE displacement from the plasma membrane (FAK-enriched fractions) to caveolin-1-enriched vesicles in vivo. (I) Western blot analysis (top) of the C1 fragment of PrP (C1) and full-length PrP (native) in cerebellar extracts from SHAM and 22L-infected mice infused or not with Y-27632 and the ratio (bottom) of C1/native full-length PrP. Note that mouse infection with 22L strain is associated with decreased PrP α-cleavage at the expense of PrP β-cleavage that generates C2 fragment [68, 69]. n = 5 for each condition. (J) ROCK immunoprecipitation followed by PDK1 western blotting in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. Values are the mean ± s.e.m. * P < 0.01 versus SHAM mice. ** P < 0.01 versus 22L-infected mice. # P < 0.05 versus SHAM mice ## P < 0.05 versus 22L-infected mice.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4524729&req=5

ppat.1005073.g006: ROCK inhibition with Y-27632 attenuates prion disease in mice.(A) Cerebellar immunoperoxidase staining to visualize PrPSc deposition in the cerebellar cortex (CBCX) and deep cerebellar nuclei (DCN) of 22L-infected mice, Scale bars, 100 μm. (B) Immunoperoxidase staining to visualize phosphorylated cofilin on Ser3 in CBCX and DCN of mock-inoculated (SHAM) and 22L-infected mice. Scale bars, 100 μm. (C) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in 22L-infected mice infused or not with Y-27632 versus SHAM mice. n = 4 in triplicate. (D) Survival curves of SHAM and 22L-inoculated mice via the intracerebellar route (i.c.b.) infused or not with the ROCK inhibitor Y-27632 by intraperitoneal injection (i.p.) starting at 130 days after infection (5 mg per kg body weight per day; 0.25 μl h-1). n = 10 mice per group. (E) Static rod test between 130 and 160 days after infection in 22L-infected mice treated with Y-27632. n = 10 mice per group. (F) Left, Western-blot for proteinase K-resistant PrPSc in brain extracts from SHAM and 22L-infected mice infused or not with Y-27632. Right, post-mortem quantification of proteinase K-resistant PrPSc in brains of 22L-infected mice treated or not with Y-27632. n = 10 for each condition. (G) PDK1 activity in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. (H) Immunoblot analysis of sucrose gradient fractions of cerebellar extracts of SHAM mice and 22L-infected mice infused or not with Y-27632 to assess TACE displacement from the plasma membrane (FAK-enriched fractions) to caveolin-1-enriched vesicles in vivo. (I) Western blot analysis (top) of the C1 fragment of PrP (C1) and full-length PrP (native) in cerebellar extracts from SHAM and 22L-infected mice infused or not with Y-27632 and the ratio (bottom) of C1/native full-length PrP. Note that mouse infection with 22L strain is associated with decreased PrP α-cleavage at the expense of PrP β-cleavage that generates C2 fragment [68, 69]. n = 5 for each condition. (J) ROCK immunoprecipitation followed by PDK1 western blotting in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. Values are the mean ± s.e.m. * P < 0.01 versus SHAM mice. ** P < 0.01 versus 22L-infected mice. # P < 0.05 versus SHAM mice ## P < 0.05 versus 22L-infected mice.

Mentions: In adult C57BL/6J mice inoculated with the mouse-adapted scrapie strain 22L via the intracerebellar route [44] and sacrificed at 130 days post infection (dpi) just before the symptomatic phase, a marked increase in phosphorylated cofilin immunostaining that matched with PrPSc deposition was observed in the brain of prion-infected mice in both the cerebellar cortex (CBCX) and deep cerebellar nuclei (DCN) compared to uninfected animals (SHAM) (Fig 6A and 6B). Western-blot analyses of cerebellum extracts indicated a ~2 to 3-fold increased level of phosphorylated cofilin in 22L-infected vs. SHAM mice (Fig 6C).


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 inhibition with Y-27632 attenuates prion disease in mice.(A) Cerebellar immunoperoxidase staining to visualize PrPSc deposition in the cerebellar cortex (CBCX) and deep cerebellar nuclei (DCN) of 22L-infected mice, Scale bars, 100 μm. (B) Immunoperoxidase staining to visualize phosphorylated cofilin on Ser3 in CBCX and DCN of mock-inoculated (SHAM) and 22L-infected mice. Scale bars, 100 μm. (C) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in 22L-infected mice infused or not with Y-27632 versus SHAM mice. n = 4 in triplicate. (D) Survival curves of SHAM and 22L-inoculated mice via the intracerebellar route (i.c.b.) infused or not with the ROCK inhibitor Y-27632 by intraperitoneal injection (i.p.) starting at 130 days after infection (5 mg per kg body weight per day; 0.25 μl h-1). n = 10 mice per group. (E) Static rod test between 130 and 160 days after infection in 22L-infected mice treated with Y-27632. n = 10 mice per group. (F) Left, Western-blot for proteinase K-resistant PrPSc in brain extracts from SHAM and 22L-infected mice infused or not with Y-27632. Right, post-mortem quantification of proteinase K-resistant PrPSc in brains of 22L-infected mice treated or not with Y-27632. n = 10 for each condition. (G) PDK1 activity in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. (H) Immunoblot analysis of sucrose gradient fractions of cerebellar extracts of SHAM mice and 22L-infected mice infused or not with Y-27632 to assess TACE displacement from the plasma membrane (FAK-enriched fractions) to caveolin-1-enriched vesicles in vivo. (I) Western blot analysis (top) of the C1 fragment of PrP (C1) and full-length PrP (native) in cerebellar extracts from SHAM and 22L-infected mice infused or not with Y-27632 and the ratio (bottom) of C1/native full-length PrP. Note that mouse infection with 22L strain is associated with decreased PrP α-cleavage at the expense of PrP β-cleavage that generates C2 fragment [68, 69]. n = 5 for each condition. (J) ROCK immunoprecipitation followed by PDK1 western blotting in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. Values are the mean ± s.e.m. * P < 0.01 versus SHAM mice. ** P < 0.01 versus 22L-infected mice. # P < 0.05 versus SHAM mice ## P < 0.05 versus 22L-infected mice.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524729&req=5

ppat.1005073.g006: ROCK inhibition with Y-27632 attenuates prion disease in mice.(A) Cerebellar immunoperoxidase staining to visualize PrPSc deposition in the cerebellar cortex (CBCX) and deep cerebellar nuclei (DCN) of 22L-infected mice, Scale bars, 100 μm. (B) Immunoperoxidase staining to visualize phosphorylated cofilin on Ser3 in CBCX and DCN of mock-inoculated (SHAM) and 22L-infected mice. Scale bars, 100 μm. (C) Western blot and histogram quantifications for phosphorylated cofilin on Ser3 in 22L-infected mice infused or not with Y-27632 versus SHAM mice. n = 4 in triplicate. (D) Survival curves of SHAM and 22L-inoculated mice via the intracerebellar route (i.c.b.) infused or not with the ROCK inhibitor Y-27632 by intraperitoneal injection (i.p.) starting at 130 days after infection (5 mg per kg body weight per day; 0.25 μl h-1). n = 10 mice per group. (E) Static rod test between 130 and 160 days after infection in 22L-infected mice treated with Y-27632. n = 10 mice per group. (F) Left, Western-blot for proteinase K-resistant PrPSc in brain extracts from SHAM and 22L-infected mice infused or not with Y-27632. Right, post-mortem quantification of proteinase K-resistant PrPSc in brains of 22L-infected mice treated or not with Y-27632. n = 10 for each condition. (G) PDK1 activity in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. (H) Immunoblot analysis of sucrose gradient fractions of cerebellar extracts of SHAM mice and 22L-infected mice infused or not with Y-27632 to assess TACE displacement from the plasma membrane (FAK-enriched fractions) to caveolin-1-enriched vesicles in vivo. (I) Western blot analysis (top) of the C1 fragment of PrP (C1) and full-length PrP (native) in cerebellar extracts from SHAM and 22L-infected mice infused or not with Y-27632 and the ratio (bottom) of C1/native full-length PrP. Note that mouse infection with 22L strain is associated with decreased PrP α-cleavage at the expense of PrP β-cleavage that generates C2 fragment [68, 69]. n = 5 for each condition. (J) ROCK immunoprecipitation followed by PDK1 western blotting in cerebellar extracts of 22L-infected mice treated or not with Y-27632 versus SHAM mice. n = 9 for each condition. Values are the mean ± s.e.m. * P < 0.01 versus SHAM mice. ** P < 0.01 versus 22L-infected mice. # P < 0.05 versus SHAM mice ## P < 0.05 versus 22L-infected mice.
Mentions: In adult C57BL/6J mice inoculated with the mouse-adapted scrapie strain 22L via the intracerebellar route [44] and sacrificed at 130 days post infection (dpi) just before the symptomatic phase, a marked increase in phosphorylated cofilin immunostaining that matched with PrPSc deposition was observed in the brain of prion-infected mice in both the cerebellar cortex (CBCX) and deep cerebellar nuclei (DCN) compared to uninfected animals (SHAM) (Fig 6A and 6B). Western-blot analyses of cerebellum extracts indicated a ~2 to 3-fold increased level of phosphorylated cofilin in 22L-infected vs. SHAM mice (Fig 6C).

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