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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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Rotarod performance and histological analyses of quadriceps in WT and VCPR155H/+ mice treated with rapamycin or chloroquine.Rotarod analysis of (A) rapamycin-treated and (B) chloroquine-treated 20-month old VCPR155H/+ and WT versus control animals at 2 week intervals. Histological analysis by H&E staining of quadriceps pathology from 20-month old control and (C-F) rapamycin- and (G-J) chloroquine-treated VCPR155H/+ and WT mice. White arrows point to overall improvement in the number of centrally located nuclei, reduced vacuoles, and an amelioration in the quadriceps fiber size and architecture. Black arrows point to worsened muscle pathology with increased vacuoles, interstitial space, and angulated fibers. The number of mice analyzed per experiment is 6–8.
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pone.0122888.g001: Rotarod performance and histological analyses of quadriceps in WT and VCPR155H/+ mice treated with rapamycin or chloroquine.Rotarod analysis of (A) rapamycin-treated and (B) chloroquine-treated 20-month old VCPR155H/+ and WT versus control animals at 2 week intervals. Histological analysis by H&E staining of quadriceps pathology from 20-month old control and (C-F) rapamycin- and (G-J) chloroquine-treated VCPR155H/+ and WT mice. White arrows point to overall improvement in the number of centrally located nuclei, reduced vacuoles, and an amelioration in the quadriceps fiber size and architecture. Black arrows point to worsened muscle pathology with increased vacuoles, interstitial space, and angulated fibers. The number of mice analyzed per experiment is 6–8.

Mentions: Our VCPR155H/+ mouse model demonstrates progressive muscle weakness approximately at the age of 6 months with vacuolization of myofibrils and centrally located nuclei, as well as cytoplasmic accumulation of TDP-43. Thus, to determine the effects of rapamycin in vivo, VCPR155H/+ and WT animals (n = 10/group) were treated with rapamycin (i.p.) at 3mg/kg/body weight and followed over a period of 8 weeks where Rotarod performance measurements were obtained at 2-week intervals. Analysis of rapamycin-treated VCPR155H/+ animals depicted a significantly improved performance in their latency to fall (seconds) off the Rotarod versus their untreated littermates at 2-, 4-, and 6-week intervals (Fig 1A). However, no significant trend was observed in Rotarod performance levels in the VCPR155H/+ animals treated with chloroquine versus their untreated littermates at 2-, 4-, and 6-week intervals (Fig 1B).


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)

Rotarod performance and histological analyses of quadriceps in WT and VCPR155H/+ mice treated with rapamycin or chloroquine.Rotarod analysis of (A) rapamycin-treated and (B) chloroquine-treated 20-month old VCPR155H/+ and WT versus control animals at 2 week intervals. Histological analysis by H&E staining of quadriceps pathology from 20-month old control and (C-F) rapamycin- and (G-J) chloroquine-treated VCPR155H/+ and WT mice. White arrows point to overall improvement in the number of centrally located nuclei, reduced vacuoles, and an amelioration in the quadriceps fiber size and architecture. Black arrows point to worsened muscle pathology with increased vacuoles, interstitial space, and angulated fibers. The number of mice analyzed per experiment is 6–8.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122888.g001: Rotarod performance and histological analyses of quadriceps in WT and VCPR155H/+ mice treated with rapamycin or chloroquine.Rotarod analysis of (A) rapamycin-treated and (B) chloroquine-treated 20-month old VCPR155H/+ and WT versus control animals at 2 week intervals. Histological analysis by H&E staining of quadriceps pathology from 20-month old control and (C-F) rapamycin- and (G-J) chloroquine-treated VCPR155H/+ and WT mice. White arrows point to overall improvement in the number of centrally located nuclei, reduced vacuoles, and an amelioration in the quadriceps fiber size and architecture. Black arrows point to worsened muscle pathology with increased vacuoles, interstitial space, and angulated fibers. The number of mice analyzed per experiment is 6–8.
Mentions: Our VCPR155H/+ mouse model demonstrates progressive muscle weakness approximately at the age of 6 months with vacuolization of myofibrils and centrally located nuclei, as well as cytoplasmic accumulation of TDP-43. Thus, to determine the effects of rapamycin in vivo, VCPR155H/+ and WT animals (n = 10/group) were treated with rapamycin (i.p.) at 3mg/kg/body weight and followed over a period of 8 weeks where Rotarod performance measurements were obtained at 2-week intervals. Analysis of rapamycin-treated VCPR155H/+ animals depicted a significantly improved performance in their latency to fall (seconds) off the Rotarod versus their untreated littermates at 2-, 4-, and 6-week intervals (Fig 1A). However, no significant trend was observed in Rotarod performance levels in the VCPR155H/+ animals treated with chloroquine versus their untreated littermates at 2-, 4-, and 6-week intervals (Fig 1B).

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