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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 autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 subjects’ myoblasts (A) untreated, (B) 10μM rapamycin-treated and (C) 10μM chloroquine-treated stained with TUNEL. VCP patients’ 421/07 myoblasts (D) untreated, (E) 10μM rapamycin-treated and (F) 10μM chloroquine-treated stained with TUNEL. Scale bar represents 100 μM. Data represents triplicate studies. (G) Percentage of TUNEL+ cells in untreated, rapamycin- and chloroquine-treated control and VCP patients’ myoblasts. Data represents triplicate studies. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.
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pone.0122888.g006: TUNEL analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 subjects’ myoblasts (A) untreated, (B) 10μM rapamycin-treated and (C) 10μM chloroquine-treated stained with TUNEL. VCP patients’ 421/07 myoblasts (D) untreated, (E) 10μM rapamycin-treated and (F) 10μM chloroquine-treated stained with TUNEL. Scale bar represents 100 μM. Data represents triplicate studies. (G) Percentage of TUNEL+ cells in untreated, rapamycin- and chloroquine-treated control and VCP patients’ myoblasts. Data represents triplicate studies. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.

Mentions: To determine apoptosis in control and patient myoblasts upon treatments with autophagy-modifying drugs, we performed TUNEL assays (Fig 6). Control 353/04 myoblasts did not show any significant differences under untreated or rapamycin-treated conditions, however, showed increased cell death after chloroquine treatment (Fig 6A–6C). Remarkably, there were fewer TUNEL+ cells after rapamycin treatment in VCP patients’ 421/07 myoblasts as compared to increased cell death after chloroquine treatment (Fig 6D–6F). Quantification of TUNEL staining after autophagy-modifying treatments in control versus VCP patients’ myoblasts (Fig 6G).


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 autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 subjects’ myoblasts (A) untreated, (B) 10μM rapamycin-treated and (C) 10μM chloroquine-treated stained with TUNEL. VCP patients’ 421/07 myoblasts (D) untreated, (E) 10μM rapamycin-treated and (F) 10μM chloroquine-treated stained with TUNEL. Scale bar represents 100 μM. Data represents triplicate studies. (G) Percentage of TUNEL+ cells in untreated, rapamycin- and chloroquine-treated control and VCP patients’ myoblasts. Data represents triplicate studies. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122888.g006: TUNEL analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 subjects’ myoblasts (A) untreated, (B) 10μM rapamycin-treated and (C) 10μM chloroquine-treated stained with TUNEL. VCP patients’ 421/07 myoblasts (D) untreated, (E) 10μM rapamycin-treated and (F) 10μM chloroquine-treated stained with TUNEL. Scale bar represents 100 μM. Data represents triplicate studies. (G) Percentage of TUNEL+ cells in untreated, rapamycin- and chloroquine-treated control and VCP patients’ myoblasts. Data represents triplicate studies. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.
Mentions: To determine apoptosis in control and patient myoblasts upon treatments with autophagy-modifying drugs, we performed TUNEL assays (Fig 6). Control 353/04 myoblasts did not show any significant differences under untreated or rapamycin-treated conditions, however, showed increased cell death after chloroquine treatment (Fig 6A–6C). Remarkably, there were fewer TUNEL+ cells after rapamycin treatment in VCP patients’ 421/07 myoblasts as compared to increased cell death after chloroquine treatment (Fig 6D–6F). Quantification of TUNEL staining after autophagy-modifying treatments in control versus VCP patients’ myoblasts (Fig 6G).

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