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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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Immunohistochemical analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 (A) untreated, (B) 10μM rapamycin-treated myoblasts, (C) 10μM chloroquine-treated myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. VCP patient 421/07 (D) untreated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43), (E) 10μM rapamycin-treated myoblasts (arrows indicated decreased expression levels of p62/SQSTM1, LC3-I/II and nuclear TDP-43), (F) 10μM chloroquine-treated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43) myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. Scale bar represents 100 μM. Data represents triplicate studies.
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pone.0122888.g005: Immunohistochemical analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 (A) untreated, (B) 10μM rapamycin-treated myoblasts, (C) 10μM chloroquine-treated myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. VCP patient 421/07 (D) untreated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43), (E) 10μM rapamycin-treated myoblasts (arrows indicated decreased expression levels of p62/SQSTM1, LC3-I/II and nuclear TDP-43), (F) 10μM chloroquine-treated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43) myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. Scale bar represents 100 μM. Data represents triplicate studies.

Mentions: Previous studies by our group have demonstrated VCP mutations in patient myoblasts causing abnormal vacuolization, autophagy and cell fusion, and increased apoptosis. To explore the in vitro effects of rapamycin and chloroquine, we treated VCP patient myoblasts (421/07) with 10μM rapamycin and stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel), and TDP-43 (lower panel) antibodies for 24 hours (Fig 5E). Similarly, we treated VCP patient myoblasts (421/07) with chloroquine and stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel), and TDP-43 (lower panel) antibodies for 24 hours (Fig 5F). Arrows point to increased expression levels of p62/SQSTM1, LC3-I/II, and TDP-43 (Fig 5D–5F). Overall, rapamycin treatment showed an improvement in the autophagy markers p62/SQSTM1 and LC3-I/II (Fig 5E), while myoblasts treated with chloroquine depicted an increased expression (Fig 5F) of autophagy markers as compared to controls (Fig 5A–5C).


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)

Immunohistochemical analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 (A) untreated, (B) 10μM rapamycin-treated myoblasts, (C) 10μM chloroquine-treated myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. VCP patient 421/07 (D) untreated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43), (E) 10μM rapamycin-treated myoblasts (arrows indicated decreased expression levels of p62/SQSTM1, LC3-I/II and nuclear TDP-43), (F) 10μM chloroquine-treated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43) myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. Scale bar represents 100 μM. Data represents triplicate studies.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122888.g005: Immunohistochemical analyses of autophagy signaling cascade in the patients’ myoblasts with VCP disease treated with either rapamycin or chloroquine.Control 353/04 (A) untreated, (B) 10μM rapamycin-treated myoblasts, (C) 10μM chloroquine-treated myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. VCP patient 421/07 (D) untreated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43), (E) 10μM rapamycin-treated myoblasts (arrows indicated decreased expression levels of p62/SQSTM1, LC3-I/II and nuclear TDP-43), (F) 10μM chloroquine-treated (white arrows represent increased p62/SQSTM1, LC3-I/II and mislocalized TDP-43) myoblasts stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel) and TDP-43 (lower panel) antibodies for 24 hours. Scale bar represents 100 μM. Data represents triplicate studies.
Mentions: Previous studies by our group have demonstrated VCP mutations in patient myoblasts causing abnormal vacuolization, autophagy and cell fusion, and increased apoptosis. To explore the in vitro effects of rapamycin and chloroquine, we treated VCP patient myoblasts (421/07) with 10μM rapamycin and stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel), and TDP-43 (lower panel) antibodies for 24 hours (Fig 5E). Similarly, we treated VCP patient myoblasts (421/07) with chloroquine and stained with p62/SQSTM1 (upper panel), LC3-I/II (middle panel), and TDP-43 (lower panel) antibodies for 24 hours (Fig 5F). Arrows point to increased expression levels of p62/SQSTM1, LC3-I/II, and TDP-43 (Fig 5D–5F). Overall, rapamycin treatment showed an improvement in the autophagy markers p62/SQSTM1 and LC3-I/II (Fig 5E), while myoblasts treated with chloroquine depicted an increased expression (Fig 5F) of autophagy markers as compared to controls (Fig 5A–5C).

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