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Neuroprotective effects of calmodulin peptide 76-121aa: disruption of calmodulin binding to mutant huntingtin.

Dudek NL, Dai Y, Muma NA - Brain Pathol. (2009)

Bottom Line: Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II.In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein.These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago School of Medicine, Maywood, IL, USA.

ABSTRACT
Huntington's disease (HD) is a neurodegenerative disease caused by mutant huntingtin protein containing an expanded polyglutamine tract, which may cause abnormal protein-protein interactions such as increased association with calmodulin (CaM). We previously demonstrated in HEK293 cells that a peptide containing amino acids 76-121 of CaM (CaM-peptide) interrupted the interaction between CaM and mutant huntingtin, reduced mutant huntingtin-induced cytotoxicity and reduced transglutaminase (TG)-modified mutant huntingtin. We now report that adeno-associated virus (AAV)-mediated expression of CaM-peptide in differentiated neuroblastoma SH-SY5Y cells, stably expressing an N-terminal fragment of huntingtin containing 148 glutamine repeats, significantly decreases the amount of TG-modified huntingtin and attenuates cytotoxicity. Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II. In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein. These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

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AAV infection (MOI 50) of differentiated neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. A. Western blot analysis of human neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. SH-SY5Y cells were transfected with N-terminal mutant huntingtin with an expanded polyglutamine repeat (SHSY5Y-htt-N63-148Q cells) and were selected based on their resistance to blasticidin. Blot was probed for N-terminal mutant huntingtin (upper) and actin (lower). B. Non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells were treated with 10 µM retinoic acid for 4 days and then infected with either AAV-CaM-peptide, AAV-scram-CaM-peptide, or AAV-GFP (MOI = 50). Forty-eight hours post-infection cells were assessed for percent AAV-mediated GFP expression by flow cytometry. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; non-htt-148Q = SH-SY5Y cells not expressing mutant huntingtin.
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fig02: AAV infection (MOI 50) of differentiated neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. A. Western blot analysis of human neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. SH-SY5Y cells were transfected with N-terminal mutant huntingtin with an expanded polyglutamine repeat (SHSY5Y-htt-N63-148Q cells) and were selected based on their resistance to blasticidin. Blot was probed for N-terminal mutant huntingtin (upper) and actin (lower). B. Non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells were treated with 10 µM retinoic acid for 4 days and then infected with either AAV-CaM-peptide, AAV-scram-CaM-peptide, or AAV-GFP (MOI = 50). Forty-eight hours post-infection cells were assessed for percent AAV-mediated GFP expression by flow cytometry. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; non-htt-148Q = SH-SY5Y cells not expressing mutant huntingtin.

Mentions: Next, we developed human neuroblastoma SH-SY5Y cell lines that stably express N-terminal mutant huntingtin. Expression levels of each cell line were estimated by Western blot analysis, and cell lines that showed moderate and similar expression were selected (Figure 2A). To further investigate the viral infection procedure, we used flow cytometry to quantitatively determine if an MOI of 50 would result in similar levels of infection for all three AAVs (AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP) in differentiated SH-SY5Y cells not expressing mutant huntingtin (non-htt-SHSY5Y cells) and in differentiated SH-SY5Y cells that stably express mutant huntingtin (SHSY5Y-htt-N63-148Q cells). Non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells were treated with 10 µM retinoic acid for 4 days to induce differentiation. Then, cells were infected with either AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP (MOI = 50). Forty-eight hours post-infection, cells were assessed for GFP expression by flow cytometry. All three viruses infected differentiated non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells with similar efficiencies (Figure 2B). However, these values were lower than previously measured for this MOI, perhaps caused by conditions associated with neuronal differentiation.


Neuroprotective effects of calmodulin peptide 76-121aa: disruption of calmodulin binding to mutant huntingtin.

Dudek NL, Dai Y, Muma NA - Brain Pathol. (2009)

AAV infection (MOI 50) of differentiated neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. A. Western blot analysis of human neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. SH-SY5Y cells were transfected with N-terminal mutant huntingtin with an expanded polyglutamine repeat (SHSY5Y-htt-N63-148Q cells) and were selected based on their resistance to blasticidin. Blot was probed for N-terminal mutant huntingtin (upper) and actin (lower). B. Non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells were treated with 10 µM retinoic acid for 4 days and then infected with either AAV-CaM-peptide, AAV-scram-CaM-peptide, or AAV-GFP (MOI = 50). Forty-eight hours post-infection cells were assessed for percent AAV-mediated GFP expression by flow cytometry. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; non-htt-148Q = SH-SY5Y cells not expressing mutant huntingtin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2805873&req=5

fig02: AAV infection (MOI 50) of differentiated neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. A. Western blot analysis of human neuroblastoma SH-SY5Y cells stably expressing N-terminal mutant huntingtin. SH-SY5Y cells were transfected with N-terminal mutant huntingtin with an expanded polyglutamine repeat (SHSY5Y-htt-N63-148Q cells) and were selected based on their resistance to blasticidin. Blot was probed for N-terminal mutant huntingtin (upper) and actin (lower). B. Non-htt-SHSY5Y cells and SHSY5Y-htt-N63-148Q cells were treated with 10 µM retinoic acid for 4 days and then infected with either AAV-CaM-peptide, AAV-scram-CaM-peptide, or AAV-GFP (MOI = 50). Forty-eight hours post-infection cells were assessed for percent AAV-mediated GFP expression by flow cytometry. Abbreviations: AAV = adeno-associated virus; CaM = calmodulin; GFP = green fluorescent protein; non-htt-148Q = SH-SY5Y cells not expressing mutant huntingtin.
Mentions: Next, we developed human neuroblastoma SH-SY5Y cell lines that stably express N-terminal mutant huntingtin. Expression levels of each cell line were estimated by Western blot analysis, and cell lines that showed moderate and similar expression were selected (Figure 2A). To further investigate the viral infection procedure, we used flow cytometry to quantitatively determine if an MOI of 50 would result in similar levels of infection for all three AAVs (AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP) in differentiated SH-SY5Y cells not expressing mutant huntingtin (non-htt-SHSY5Y cells) and in differentiated SH-SY5Y cells that stably express mutant huntingtin (SHSY5Y-htt-N63-148Q cells). Non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells were treated with 10 µM retinoic acid for 4 days to induce differentiation. Then, cells were infected with either AAV-CaM-peptide + GFP, AAV-scram-CaM-peptide + GFP or AAV-GFP (MOI = 50). Forty-eight hours post-infection, cells were assessed for GFP expression by flow cytometry. All three viruses infected differentiated non-htt-SHSY5Y and SHSY5Y-htt-N63-148Q cells with similar efficiencies (Figure 2B). However, these values were lower than previously measured for this MOI, perhaps caused by conditions associated with neuronal differentiation.

Bottom Line: Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II.In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein.These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago School of Medicine, Maywood, IL, USA.

ABSTRACT
Huntington's disease (HD) is a neurodegenerative disease caused by mutant huntingtin protein containing an expanded polyglutamine tract, which may cause abnormal protein-protein interactions such as increased association with calmodulin (CaM). We previously demonstrated in HEK293 cells that a peptide containing amino acids 76-121 of CaM (CaM-peptide) interrupted the interaction between CaM and mutant huntingtin, reduced mutant huntingtin-induced cytotoxicity and reduced transglutaminase (TG)-modified mutant huntingtin. We now report that adeno-associated virus (AAV)-mediated expression of CaM-peptide in differentiated neuroblastoma SH-SY5Y cells, stably expressing an N-terminal fragment of huntingtin containing 148 glutamine repeats, significantly decreases the amount of TG-modified huntingtin and attenuates cytotoxicity. Importantly, the effect of the CaM-peptide shows selectivity, such that total TG activity is not significantly altered by expression of CaM-peptide nor is the activity of another CaM-dependent enzyme, CaM kinase II. In vitro, recombinant exon 1 of huntingtin with 44 glutamines (htt-exon1-44Q) binds to CaM-agarose; the addition of 10 microM of CaM-peptide significantly decreases the interaction of htt-exon1-44Q and CaM but not the binding between CaM and calcineurin, another CaM-binding protein. These data support the hypothesis that CaM regulates TG-catalyzed modifications of mutant huntingtin and that specific and selective disruption of the CaM-huntingtin interaction is potentially a new target for therapeutic intervention in HD.

Show MeSH
Related in: MedlinePlus