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Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

Nalbandian A, Llewellyn KJ, Nguyen C, Yazdi PG, Kimonis VE - PLoS ONE (2015)

Bottom Line: Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux.Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers.Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Genetics and Metabolism, University of California, Irvine, California, United States of America; Sue and Bill Gross Stem Cell Center, University of California, Irvine, California, United States of America.

ABSTRACT
Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

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TUNEL analyses of quadriceps in rapamycin- and chloroquine-treated VCPR155H/+ and WT mice.TUNEL staining of quadriceps muscles from (A,B) control untreated (C,D) rapamycin-treated and (E,F) chloroquine-treated WT and VCPR155H/+ animals at 20 months of age. (G) Quantification of TUNEL+ cells in control and treated VCPR155H/+ and WT animals. Arrows point to TUNEL+ cells indicating cell death. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test. The number of animals used was n = 8-10/group.
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pone.0122888.g004: TUNEL analyses of quadriceps in rapamycin- and chloroquine-treated VCPR155H/+ and WT mice.TUNEL staining of quadriceps muscles from (A,B) control untreated (C,D) rapamycin-treated and (E,F) chloroquine-treated WT and VCPR155H/+ animals at 20 months of age. (G) Quantification of TUNEL+ cells in control and treated VCPR155H/+ and WT animals. Arrows point to TUNEL+ cells indicating cell death. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test. The number of animals used was n = 8-10/group.

Mentions: To examine the effect of autophagy-modifying drugs on apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed on the quadriceps sections of treated and control WT and VCPR155H/+ mice. Rapamycin-treated muscle fibers of the VCPR155H/+ mice displayed reduced levels of apoptosis, as there were significantly fewer TUNEL positive cells as compared to WT littermates (Fig 4A–4D). However, no difference was observed in cell death in the chloroquine-treated WT and VCPR155H/+ quadriceps (Fig 4A, 4B, 4E, and 4F) as compared to untreated control littermates. Quantification of TUNEL+ cells depicting 12% cell death in vehicle control WT, 31% in vehicle control VCPR155H/+, 10% in WT and 15% in rapamycin-treated mice, and 18% in WT and 29% in VCPR155H/+ chloroquine-treated mice (Fig 4G). Statistical significance (p<0.005) was observed between the control VCPR155H/+ and rapamycin-treated VCPR155H/+ mice (Fig 4G).


Rapamycin and chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show promising results in valosin containing protein multisystem proteinopathy.

Nalbandian A, Llewellyn KJ, Nguyen C, Yazdi PG, Kimonis VE - PLoS ONE (2015)

TUNEL analyses of quadriceps in rapamycin- and chloroquine-treated VCPR155H/+ and WT mice.TUNEL staining of quadriceps muscles from (A,B) control untreated (C,D) rapamycin-treated and (E,F) chloroquine-treated WT and VCPR155H/+ animals at 20 months of age. (G) Quantification of TUNEL+ cells in control and treated VCPR155H/+ and WT animals. Arrows point to TUNEL+ cells indicating cell death. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test. The number of animals used was n = 8-10/group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4401571&req=5

pone.0122888.g004: TUNEL analyses of quadriceps in rapamycin- and chloroquine-treated VCPR155H/+ and WT mice.TUNEL staining of quadriceps muscles from (A,B) control untreated (C,D) rapamycin-treated and (E,F) chloroquine-treated WT and VCPR155H/+ animals at 20 months of age. (G) Quantification of TUNEL+ cells in control and treated VCPR155H/+ and WT animals. Arrows point to TUNEL+ cells indicating cell death. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test. The number of animals used was n = 8-10/group.
Mentions: To examine the effect of autophagy-modifying drugs on apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed on the quadriceps sections of treated and control WT and VCPR155H/+ mice. Rapamycin-treated muscle fibers of the VCPR155H/+ mice displayed reduced levels of apoptosis, as there were significantly fewer TUNEL positive cells as compared to WT littermates (Fig 4A–4D). However, no difference was observed in cell death in the chloroquine-treated WT and VCPR155H/+ quadriceps (Fig 4A, 4B, 4E, and 4F) as compared to untreated control littermates. Quantification of TUNEL+ cells depicting 12% cell death in vehicle control WT, 31% in vehicle control VCPR155H/+, 10% in WT and 15% in rapamycin-treated mice, and 18% in WT and 29% in VCPR155H/+ chloroquine-treated mice (Fig 4G). Statistical significance (p<0.005) was observed between the control VCPR155H/+ and rapamycin-treated VCPR155H/+ mice (Fig 4G).

Bottom Line: Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux.Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers.Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Genetics and Metabolism, University of California, Irvine, California, United States of America; Sue and Bill Gross Stem Cell Center, University of California, Irvine, California, United States of America.

ABSTRACT
Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can now be included as disorders that can potentially be ameliorated by rapalogs.

Show MeSH
Related in: MedlinePlus