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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 quadriceps of VCPR155H/+ and WT mice treated with autophagy-modifying drugs.Quadriceps muscles from (A,B) control, (C,D) rapamycin- and (E,F) chloroquine-treated 20-month old WT and VCPR155H/+ mice were stained with anti-ubiquitin, p62/SQSTM1, LC3-I/II, and TDP-43/ubiquitin specific antibodies, respectively (shown by arrows). Cells’ nuclei were stained with DAPI (Magnification: 630X). Scale bar represents 100 μM. (G) Western blot expression analysis of autophagy proteins including ubiquitin, optineurin (OPTN), p62/SQSTM1, VCP, LC3-I/II, TDP-43, and mTOR pathway proteins: mTOR and p70S6K. Beta actin was used as a positive control. The number of mice analyzed per experiment is 6–8.
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pone.0122888.g002: Immunohistochemical analyses of autophagy signaling cascade in the quadriceps of VCPR155H/+ and WT mice treated with autophagy-modifying drugs.Quadriceps muscles from (A,B) control, (C,D) rapamycin- and (E,F) chloroquine-treated 20-month old WT and VCPR155H/+ mice were stained with anti-ubiquitin, p62/SQSTM1, LC3-I/II, and TDP-43/ubiquitin specific antibodies, respectively (shown by arrows). Cells’ nuclei were stained with DAPI (Magnification: 630X). Scale bar represents 100 μM. (G) Western blot expression analysis of autophagy proteins including ubiquitin, optineurin (OPTN), p62/SQSTM1, VCP, LC3-I/II, TDP-43, and mTOR pathway proteins: mTOR and p70S6K. Beta actin was used as a positive control. The number of mice analyzed per experiment is 6–8.

Mentions: To elucidate the effects of rapamycin on the autophagy signaling pathway, we analyzed the autophagy intermediates. Autophagy flux was monitored by detection of endogenous LC3-I/II modification, ubiquitin-positive inclusions, and p62/SQSTM1 and optineurin aggregates. In comparison to the control WT and VCPR155H/+ mice (Fig 2A and 2B), the rapamycin-treated VCPR155H/+ mice demonstrated an overall decrease in ubiquitinated proteins, a decrease in LC3-I expression followed by an increased conversion to LC3-II, and a decrease in p62/SQSTM1 and optineurin (OPTN), expression levels, suggesting an improvement of the autophagic process in comparison with the rapamycin-treated animals (Fig 2C and 2D). Furthermore, we examined the TDP-43 aggregates (nuclear to cytoplasmic translocation) in the VCPR155H/+ animals versus their WT littermates. The rapamycin-treated VCPR155H/+ mice showed more nuclear TDP-43 expression, which is suggestive of a more normal phenotype. In contrast, chloroquine-treated depicted an overall increase in these autophagy intermediates (Fig 2E and 2F). Western blot of ubiquitin, TDP-43 p62/SQSTM1, LC3-I/II and OPTN confirmed these findings (Fig 2G). Distribution levels of VCP expression were comparable in quadriceps muscle of WT and VCPR155H/+ (data not shown).


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 quadriceps of VCPR155H/+ and WT mice treated with autophagy-modifying drugs.Quadriceps muscles from (A,B) control, (C,D) rapamycin- and (E,F) chloroquine-treated 20-month old WT and VCPR155H/+ mice were stained with anti-ubiquitin, p62/SQSTM1, LC3-I/II, and TDP-43/ubiquitin specific antibodies, respectively (shown by arrows). Cells’ nuclei were stained with DAPI (Magnification: 630X). Scale bar represents 100 μM. (G) Western blot expression analysis of autophagy proteins including ubiquitin, optineurin (OPTN), p62/SQSTM1, VCP, LC3-I/II, TDP-43, and mTOR pathway proteins: mTOR and p70S6K. Beta actin was used as a positive control. The number of mice analyzed per experiment is 6–8.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122888.g002: Immunohistochemical analyses of autophagy signaling cascade in the quadriceps of VCPR155H/+ and WT mice treated with autophagy-modifying drugs.Quadriceps muscles from (A,B) control, (C,D) rapamycin- and (E,F) chloroquine-treated 20-month old WT and VCPR155H/+ mice were stained with anti-ubiquitin, p62/SQSTM1, LC3-I/II, and TDP-43/ubiquitin specific antibodies, respectively (shown by arrows). Cells’ nuclei were stained with DAPI (Magnification: 630X). Scale bar represents 100 μM. (G) Western blot expression analysis of autophagy proteins including ubiquitin, optineurin (OPTN), p62/SQSTM1, VCP, LC3-I/II, TDP-43, and mTOR pathway proteins: mTOR and p70S6K. Beta actin was used as a positive control. The number of mice analyzed per experiment is 6–8.
Mentions: To elucidate the effects of rapamycin on the autophagy signaling pathway, we analyzed the autophagy intermediates. Autophagy flux was monitored by detection of endogenous LC3-I/II modification, ubiquitin-positive inclusions, and p62/SQSTM1 and optineurin aggregates. In comparison to the control WT and VCPR155H/+ mice (Fig 2A and 2B), the rapamycin-treated VCPR155H/+ mice demonstrated an overall decrease in ubiquitinated proteins, a decrease in LC3-I expression followed by an increased conversion to LC3-II, and a decrease in p62/SQSTM1 and optineurin (OPTN), expression levels, suggesting an improvement of the autophagic process in comparison with the rapamycin-treated animals (Fig 2C and 2D). Furthermore, we examined the TDP-43 aggregates (nuclear to cytoplasmic translocation) in the VCPR155H/+ animals versus their WT littermates. The rapamycin-treated VCPR155H/+ mice showed more nuclear TDP-43 expression, which is suggestive of a more normal phenotype. In contrast, chloroquine-treated depicted an overall increase in these autophagy intermediates (Fig 2E and 2F). Western blot of ubiquitin, TDP-43 p62/SQSTM1, LC3-I/II and OPTN confirmed these findings (Fig 2G). Distribution levels of VCP expression were comparable in quadriceps muscle of WT and VCPR155H/+ (data not shown).

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