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Human adipose-derived mesenchymal stem cells as a new model of spinal and bulbar muscular atrophy.

Dossena M, Bedini G, Rusmini P, Giorgetti E, Canazza A, Tosetti V, Salsano E, Sagnelli A, Mariotti C, Gellera C, Navone SE, Marfia G, Alessandri G, Corsi F, Parati EA, Pareyson D, Poletti A - PLoS ONE (2014)

Bottom Line: These cells have the advantage to express only ARpolyQ, and not the wild type AR allele.Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs.In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Laboratory, Unit of Cerebrovascular Disease, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

ABSTRACT
Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked CAG/polyglutamine expansion motoneuron disease, in which an elongated polyglutamine tract (polyQ) in the N-terminal androgen receptor (ARpolyQ) confers toxicity to this protein. Typical markers of SBMA disease are ARpolyQ intranuclear inclusions. These are generated after the ARpolyQ binds to its endogenous ligands, which promotes AR release from chaperones, activation and nuclear translocation, but also cell toxicity. The SBMA mouse models developed so far, and used in preclinical studies, all contain an expanded CAG repeat significantly longer than that of SBMA patients. Here, we propose the use of SBMA patients adipose-derived mesenchymal stem cells (MSCs) as a new human in vitro model to study ARpolyQ toxicity. These cells have the advantage to express only ARpolyQ, and not the wild type AR allele. Therefore, we isolated and characterized adipose-derived MSCs from three SBMA patients (ADSC from Kennedy's patients, ADSCK) and three control volunteers (ADSCs). We found that both ADSCs and ADSCKs express mesenchymal antigens, even if only ADSCs can differentiate into the three typical cell lineages (adipocytes, chondrocytes and osteocytes), whereas ADSCKs, from SBMA patients, showed a lower growth potential and differentiated only into adipocyte. Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs. In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions. Considering all of this evidence, SBMA patients adipose-derived MSCs cultures should be considered an innovative in vitro human model to understand the molecular mechanisms of ARpolyQ toxicity and to test novel therapeutic approaches in SBMA.

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Biochemical behaviour of AR in ADSCs and ADSCKs.(A) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCs and ADSCKs in absence (–T) or in presence (+T) of 10 nM of testosterone for 48 hours in basal condition or after treatment with 10 µM of MG132 for 24 hours. Nuclei were stained with DAPI. Scale bar 10 µm. (B) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in the presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and Hsp70 (red). Nuclei were stained with DAPI. (C) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and ubiquitin (red). Nuclei were stained with DAPI. Panel B and C represent only the nuclear area of the cells where the aggregates were observed. The arrows indicate the ARpolyQ inclusions co-stained with Hsp70 and ubiquitin, respectively.
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pone-0112746-g004: Biochemical behaviour of AR in ADSCs and ADSCKs.(A) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCs and ADSCKs in absence (–T) or in presence (+T) of 10 nM of testosterone for 48 hours in basal condition or after treatment with 10 µM of MG132 for 24 hours. Nuclei were stained with DAPI. Scale bar 10 µm. (B) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in the presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and Hsp70 (red). Nuclei were stained with DAPI. (C) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and ubiquitin (red). Nuclei were stained with DAPI. Panel B and C represent only the nuclear area of the cells where the aggregates were observed. The arrows indicate the ARpolyQ inclusions co-stained with Hsp70 and ubiquitin, respectively.

Mentions: To evaluate whether testosterone normally induces wtAR or ARpolyQ translocation into the nuclei of ADSCs, we performed immunofluorescence analysis using an anti-AR antibody. Figure 4A shows that without testosterone both ARs were cytoplasmic; testosterone treatment (10 nM) for 48 hours induced complete wtAR translocation to the nucleus, while ARpolyQ did not completely translocate to the nucleus, as a small amount remained cytoplasmic. When we evaluated the effect of proteasome blockage on AR clearance, we found that treatment with proteasome inhibitor MG132 induced an overall increase of both wtAR and ARpolyQ levels in cytoplasm and nucleus. Interestingly, we noted that in ADSCKs the co-treatment with testosterone and MG132 induced formation of ARpolyQ nuclear inclusions in a few cells (approximately 5% of total analysed cells); the autophagy inhibitor 3-MA increased ARpolyQ levels, but did not induce intranuclear inclusions (not shown). Thus, the proteasome appears to be one of the preferential pathways to clear the misfolded fraction of ARpolyQ, capable to aggregate, present in ADSCKs. Therefore, we analysed whether MG132-induced ARpolyQ nuclear inclusions directly resulted from inhibition of degradative systems, by analysing possible sequestration of two proteins essential for proteasomal degradation. We found that testosterone- and MG132-induced nuclear inclusions sequestered the chaperone HSP70 (responsible for recognition of misfolded protein species to be degraded by ubiquitin-proteasome pathway) (Figure 4B) and were also positive for ubiquitin (Figure 4C). This suggests that some misfolded species of ARpolyQ present in ADSCKs require a functional proteasome for their clearance, even if an indirect effect can also be postulated. In fact, several stressor stimuli resulting from proteasome blockage (which limits the clearance also of other ubiquitinated proteins) are added to a system, which is possibly almost overwhelmed by the presence of mutant ARpolyQ.


Human adipose-derived mesenchymal stem cells as a new model of spinal and bulbar muscular atrophy.

Dossena M, Bedini G, Rusmini P, Giorgetti E, Canazza A, Tosetti V, Salsano E, Sagnelli A, Mariotti C, Gellera C, Navone SE, Marfia G, Alessandri G, Corsi F, Parati EA, Pareyson D, Poletti A - PLoS ONE (2014)

Biochemical behaviour of AR in ADSCs and ADSCKs.(A) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCs and ADSCKs in absence (–T) or in presence (+T) of 10 nM of testosterone for 48 hours in basal condition or after treatment with 10 µM of MG132 for 24 hours. Nuclei were stained with DAPI. Scale bar 10 µm. (B) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in the presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and Hsp70 (red). Nuclei were stained with DAPI. (C) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and ubiquitin (red). Nuclei were stained with DAPI. Panel B and C represent only the nuclear area of the cells where the aggregates were observed. The arrows indicate the ARpolyQ inclusions co-stained with Hsp70 and ubiquitin, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231043&req=5

pone-0112746-g004: Biochemical behaviour of AR in ADSCs and ADSCKs.(A) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCs and ADSCKs in absence (–T) or in presence (+T) of 10 nM of testosterone for 48 hours in basal condition or after treatment with 10 µM of MG132 for 24 hours. Nuclei were stained with DAPI. Scale bar 10 µm. (B) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in the presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and Hsp70 (red). Nuclei were stained with DAPI. (C) High resolution fluorescence microscopy analysis (63X magnification) performed on ADSCKs in presence (+T) of 10 nM of testosterone for 48 hours after treatment with 10 µM of MG132 for 24 hours. Fluorescence microscopy localization of AR (green) and ubiquitin (red). Nuclei were stained with DAPI. Panel B and C represent only the nuclear area of the cells where the aggregates were observed. The arrows indicate the ARpolyQ inclusions co-stained with Hsp70 and ubiquitin, respectively.
Mentions: To evaluate whether testosterone normally induces wtAR or ARpolyQ translocation into the nuclei of ADSCs, we performed immunofluorescence analysis using an anti-AR antibody. Figure 4A shows that without testosterone both ARs were cytoplasmic; testosterone treatment (10 nM) for 48 hours induced complete wtAR translocation to the nucleus, while ARpolyQ did not completely translocate to the nucleus, as a small amount remained cytoplasmic. When we evaluated the effect of proteasome blockage on AR clearance, we found that treatment with proteasome inhibitor MG132 induced an overall increase of both wtAR and ARpolyQ levels in cytoplasm and nucleus. Interestingly, we noted that in ADSCKs the co-treatment with testosterone and MG132 induced formation of ARpolyQ nuclear inclusions in a few cells (approximately 5% of total analysed cells); the autophagy inhibitor 3-MA increased ARpolyQ levels, but did not induce intranuclear inclusions (not shown). Thus, the proteasome appears to be one of the preferential pathways to clear the misfolded fraction of ARpolyQ, capable to aggregate, present in ADSCKs. Therefore, we analysed whether MG132-induced ARpolyQ nuclear inclusions directly resulted from inhibition of degradative systems, by analysing possible sequestration of two proteins essential for proteasomal degradation. We found that testosterone- and MG132-induced nuclear inclusions sequestered the chaperone HSP70 (responsible for recognition of misfolded protein species to be degraded by ubiquitin-proteasome pathway) (Figure 4B) and were also positive for ubiquitin (Figure 4C). This suggests that some misfolded species of ARpolyQ present in ADSCKs require a functional proteasome for their clearance, even if an indirect effect can also be postulated. In fact, several stressor stimuli resulting from proteasome blockage (which limits the clearance also of other ubiquitinated proteins) are added to a system, which is possibly almost overwhelmed by the presence of mutant ARpolyQ.

Bottom Line: These cells have the advantage to express only ARpolyQ, and not the wild type AR allele.Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs.In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Laboratory, Unit of Cerebrovascular Disease, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

ABSTRACT
Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked CAG/polyglutamine expansion motoneuron disease, in which an elongated polyglutamine tract (polyQ) in the N-terminal androgen receptor (ARpolyQ) confers toxicity to this protein. Typical markers of SBMA disease are ARpolyQ intranuclear inclusions. These are generated after the ARpolyQ binds to its endogenous ligands, which promotes AR release from chaperones, activation and nuclear translocation, but also cell toxicity. The SBMA mouse models developed so far, and used in preclinical studies, all contain an expanded CAG repeat significantly longer than that of SBMA patients. Here, we propose the use of SBMA patients adipose-derived mesenchymal stem cells (MSCs) as a new human in vitro model to study ARpolyQ toxicity. These cells have the advantage to express only ARpolyQ, and not the wild type AR allele. Therefore, we isolated and characterized adipose-derived MSCs from three SBMA patients (ADSC from Kennedy's patients, ADSCK) and three control volunteers (ADSCs). We found that both ADSCs and ADSCKs express mesenchymal antigens, even if only ADSCs can differentiate into the three typical cell lineages (adipocytes, chondrocytes and osteocytes), whereas ADSCKs, from SBMA patients, showed a lower growth potential and differentiated only into adipocyte. Moreover, analysing AR expression on our mesenchymal cultures we found lower levels in all ADSCKs than ADSCs, possibly related to negative pressures exerted by toxic ARpolyQ in ADSCKs. In addition, with proteasome inhibition the ARpolyQ levels increased specifically in ADSCKs, inducing the formation of HSP70 and ubiquitin positive nuclear ARpolyQ inclusions. Considering all of this evidence, SBMA patients adipose-derived MSCs cultures should be considered an innovative in vitro human model to understand the molecular mechanisms of ARpolyQ toxicity and to test novel therapeutic approaches in SBMA.

Show MeSH
Related in: MedlinePlus