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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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Mitochondrial enzyme analyses of the VCPR155H/+ and WT mouse quadriceps treated with rapamycin or chloroquine.Quadriceps muscles from (A,D) vehicle control (B,E) rapamycin-treated or (C,F) chloroquine-treated animals were stained with SDH antibody to observe mitochondrial proliferation and oxidative fibers/capacity (Black arrows point to Type II dark fibers; White arrows point to lighter fibers) and (G-L) Oil Red O to observe lipid droplets in WT and VCPR155H/+ mice at 20 months of age (Magnification: 400X). Black arrows point to increased Oil Red O Staining; white arrows point to diminished Oil Red O staining. (M) Quantification of Type II oxidative fibers with autophagy-modifying drugs. The number of mice analyzed per experiment is 8–10. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.
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pone.0122888.g003: Mitochondrial enzyme analyses of the VCPR155H/+ and WT mouse quadriceps treated with rapamycin or chloroquine.Quadriceps muscles from (A,D) vehicle control (B,E) rapamycin-treated or (C,F) chloroquine-treated animals were stained with SDH antibody to observe mitochondrial proliferation and oxidative fibers/capacity (Black arrows point to Type II dark fibers; White arrows point to lighter fibers) and (G-L) Oil Red O to observe lipid droplets in WT and VCPR155H/+ mice at 20 months of age (Magnification: 400X). Black arrows point to increased Oil Red O Staining; white arrows point to diminished Oil Red O staining. (M) Quantification of Type II oxidative fibers with autophagy-modifying drugs. The number of mice analyzed per experiment is 8–10. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.

Mentions: To examine the effects of rapamycin or chloroquine administration on the mitochondrial complexes of VCPR155H/+ and WT animals, we performed mitochondrial assays. Identification of oxidative and non-oxidative fibers is used in assessing mitochondrial pathology. Compared to 20-month old WT littermates which depicted a normal “checkered” pattern, succinic dehydrogenase (SDH) staining of heterozygous VCPR155H/+ mice quadriceps revealed increased Type II fibers (dark fibers) oxidative fibers (Fig 3A–3F). VCPR155H/+ heterozygous mice treated with rapamycin revealed a decrease in Type II fibers (dark fibers) suggestive of normal mitochondrial proliferation and balanced oxidative capacity (Fig 3E). Interestingly, chloroquine had no effect on the Type II fibers (Fig 3F). Quantification of oxidative fibers with autophagy-modifying drugs is shown in Fig 3M. Recently, pregnant dams and their pups fed a lipid-enriched diet (LED) resulted in the reversal of the lethal phenotype in homozygous offspring and improved survival, motor activity, muscle pathology and the autophagy cascade suggesting that lipid supplementation may be a promising therapeutic strategy for patients with VCP-associated neurodegenerative diseases [34]. Thus, we analyzed generation of lipid granules in our mice and did not note granules in WT mice by Oil Red O staining, however, the untreated heterozygous VCPR155H/+ quadriceps muscles showed small lipid granule accumulation in a scattered pattern (as shown with black arrows) (Fig 3G–3L). Remarkably, these lipid granules were markedly reduced in the VCPR155H/+ mice treated with rapamycin (as shown with white arrows) (Fig 3K). Conversely, chloroquine treatment resulted in an accumulation of these lipid particles in the quadriceps muscles (as indicated with black arrows) (Fig 3L).


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)

Mitochondrial enzyme analyses of the VCPR155H/+ and WT mouse quadriceps treated with rapamycin or chloroquine.Quadriceps muscles from (A,D) vehicle control (B,E) rapamycin-treated or (C,F) chloroquine-treated animals were stained with SDH antibody to observe mitochondrial proliferation and oxidative fibers/capacity (Black arrows point to Type II dark fibers; White arrows point to lighter fibers) and (G-L) Oil Red O to observe lipid droplets in WT and VCPR155H/+ mice at 20 months of age (Magnification: 400X). Black arrows point to increased Oil Red O Staining; white arrows point to diminished Oil Red O staining. (M) Quantification of Type II oxidative fibers with autophagy-modifying drugs. The number of mice analyzed per experiment is 8–10. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122888.g003: Mitochondrial enzyme analyses of the VCPR155H/+ and WT mouse quadriceps treated with rapamycin or chloroquine.Quadriceps muscles from (A,D) vehicle control (B,E) rapamycin-treated or (C,F) chloroquine-treated animals were stained with SDH antibody to observe mitochondrial proliferation and oxidative fibers/capacity (Black arrows point to Type II dark fibers; White arrows point to lighter fibers) and (G-L) Oil Red O to observe lipid droplets in WT and VCPR155H/+ mice at 20 months of age (Magnification: 400X). Black arrows point to increased Oil Red O Staining; white arrows point to diminished Oil Red O staining. (M) Quantification of Type II oxidative fibers with autophagy-modifying drugs. The number of mice analyzed per experiment is 8–10. Statistical significance is denoted by *p<0.005 by Student one-tailed t-test.
Mentions: To examine the effects of rapamycin or chloroquine administration on the mitochondrial complexes of VCPR155H/+ and WT animals, we performed mitochondrial assays. Identification of oxidative and non-oxidative fibers is used in assessing mitochondrial pathology. Compared to 20-month old WT littermates which depicted a normal “checkered” pattern, succinic dehydrogenase (SDH) staining of heterozygous VCPR155H/+ mice quadriceps revealed increased Type II fibers (dark fibers) oxidative fibers (Fig 3A–3F). VCPR155H/+ heterozygous mice treated with rapamycin revealed a decrease in Type II fibers (dark fibers) suggestive of normal mitochondrial proliferation and balanced oxidative capacity (Fig 3E). Interestingly, chloroquine had no effect on the Type II fibers (Fig 3F). Quantification of oxidative fibers with autophagy-modifying drugs is shown in Fig 3M. Recently, pregnant dams and their pups fed a lipid-enriched diet (LED) resulted in the reversal of the lethal phenotype in homozygous offspring and improved survival, motor activity, muscle pathology and the autophagy cascade suggesting that lipid supplementation may be a promising therapeutic strategy for patients with VCP-associated neurodegenerative diseases [34]. Thus, we analyzed generation of lipid granules in our mice and did not note granules in WT mice by Oil Red O staining, however, the untreated heterozygous VCPR155H/+ quadriceps muscles showed small lipid granule accumulation in a scattered pattern (as shown with black arrows) (Fig 3G–3L). Remarkably, these lipid granules were markedly reduced in the VCPR155H/+ mice treated with rapamycin (as shown with white arrows) (Fig 3K). Conversely, chloroquine treatment resulted in an accumulation of these lipid particles in the quadriceps muscles (as indicated with black arrows) (Fig 3L).

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