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Blocking GluR2-GAPDH ameliorates experimental autoimmune encephalomyelitis.

Zhai D, Lee FH, D'Souza C, Su P, Zhang S, Jia Z, Zhang L, Wong AH, Liu F - Ann Clin Transl Neurol (2015)

Bottom Line: We found that protein complex composed of the GluR2 subunit of AMPA receptors and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was present at significantly higher levels in postmortem tissue from MS patients and in EAE mice, an animal model for MS.This peptide greatly improves neurological function in EAE mice, reduces neuron death, rescues demyelination, increases oligodendrocyte survival, and reduces axonal damage in the spinal cords of EAE mice.More importantly, our peptide has no direct suppressive effect on naive T-cell responses or basal neurotransmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Centre for Addiction and Mental Health Toronto, Ontario, Canada, M5T 1R8.

ABSTRACT

Objective: Multiple sclerosis (MS) is the most common disabling neurological disease of young adults. The pathophysiological mechanism of MS remains largely unknown and no cure is available. Current clinical treatments for MS modulate the immune system, with the rationale that autoimmunity is at the core of MS pathophysiology.

Methods: Experimental autoimmune encephalitis (EAE) was induced in mice with MOG35-55 and clinical scoring was performed to monitor signs of paralysis. EAE mice were injected intraperitoneally with TAT-fusion peptides daily from day 10 until day 30 after immunization, and their effects were measured at day 17 or day 30.

Results: We report a novel target for the development of MS therapy, which aimed at blocking glutamate-mediated neurotoxicity through targeting the interaction between the AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid) receptor and an interacting protein. We found that protein complex composed of the GluR2 subunit of AMPA receptors and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was present at significantly higher levels in postmortem tissue from MS patients and in EAE mice, an animal model for MS. Next, we developed a peptide that specifically disrupts the GluR2 -GAPDH complex. This peptide greatly improves neurological function in EAE mice, reduces neuron death, rescues demyelination, increases oligodendrocyte survival, and reduces axonal damage in the spinal cords of EAE mice. More importantly, our peptide has no direct suppressive effect on naive T-cell responses or basal neurotransmission.

Interpretation: The GluR2 -GAPDH complex represents a novel therapeutic target for the development of medications for MS that work through a different mechanism than existing treatments.

No MeSH data available.


Related in: MedlinePlus

GluR2–GAPDH complex in multiple sclerosis (MS). (A, B) GluR2–GAPDH complex formation is significantly increased in the plaque of multiple sclerosis (MS). Postmortem samples from control, MS plaque (plaque+) area, and MS nonplaque (plaque−) area were incubated with GluR2 antibody and the precipitated proteins were immunoblotted with GAPDH antibody or GluR2 antibody. The intensity of each protein band for GAPDH (A), GluR2 (B) from all three groups was quantified by densitometry (AIS software, Imaging Research Inc.). Results for each sample are presented as the percentage of the mean of the control samples on the same blot. (*P < 0.05, n = 8, one-way ANOVA). (C–E) GluR2–GAPDH complex formation is significantly increased in experimental autoimmune encephalitis (EAE) mice compared to sham mice. (A) Representative image of western blot analysis of GAPDH (top) and GluR2 (bottom) levels precipitated by GluR2 antibody in extract prepared from mouse spinal cord tissue were incubated with GluR2 antibody. Precipitated proteins were subject to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotted with GAPDH or GluR2 antibody. (D, E) Densitometric analysis of the level of GAPDH and GluR2. The intensity of GAPDH and GluR2 was quantified by densitometry (software: Image J, NIH). Data were analyzed by t-test. (**P < 0.01, n = 3). (F) Clinical EAE scores (mean ± SEM) over time of four groups vaccinated with MOG35-55 on day 0 and treated intraperitoneally daily with TAT-G-Gpep and TAT-G-Gpep-Sc from day 10 (arrow). Starting from day 12, P < 0.05, data were analyzed by Mann–Whitney U test.
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fig01: GluR2–GAPDH complex in multiple sclerosis (MS). (A, B) GluR2–GAPDH complex formation is significantly increased in the plaque of multiple sclerosis (MS). Postmortem samples from control, MS plaque (plaque+) area, and MS nonplaque (plaque−) area were incubated with GluR2 antibody and the precipitated proteins were immunoblotted with GAPDH antibody or GluR2 antibody. The intensity of each protein band for GAPDH (A), GluR2 (B) from all three groups was quantified by densitometry (AIS software, Imaging Research Inc.). Results for each sample are presented as the percentage of the mean of the control samples on the same blot. (*P < 0.05, n = 8, one-way ANOVA). (C–E) GluR2–GAPDH complex formation is significantly increased in experimental autoimmune encephalitis (EAE) mice compared to sham mice. (A) Representative image of western blot analysis of GAPDH (top) and GluR2 (bottom) levels precipitated by GluR2 antibody in extract prepared from mouse spinal cord tissue were incubated with GluR2 antibody. Precipitated proteins were subject to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotted with GAPDH or GluR2 antibody. (D, E) Densitometric analysis of the level of GAPDH and GluR2. The intensity of GAPDH and GluR2 was quantified by densitometry (software: Image J, NIH). Data were analyzed by t-test. (**P < 0.01, n = 3). (F) Clinical EAE scores (mean ± SEM) over time of four groups vaccinated with MOG35-55 on day 0 and treated intraperitoneally daily with TAT-G-Gpep and TAT-G-Gpep-Sc from day 10 (arrow). Starting from day 12, P < 0.05, data were analyzed by Mann–Whitney U test.

Mentions: As shown in Figure1A, co-immunoprecipitation of GAPDH by the GluR2 antibody was significantly greater in protein extracted from MS spinal cord plaques compared to both healthy regions spinal cord from MS patients and healthy control subjects (n = 8, P < 0.05). In contrast, neither the levels of directly immunoprecipitated GluR2 (Fig.1B) nor the expression of GluR2 and GAPDH was significantly different between all groups (Fig. S1A and B). These data suggest an increased level of GluR2–GAPDH complex in MS plaques but not healthy tissue.


Blocking GluR2-GAPDH ameliorates experimental autoimmune encephalomyelitis.

Zhai D, Lee FH, D'Souza C, Su P, Zhang S, Jia Z, Zhang L, Wong AH, Liu F - Ann Clin Transl Neurol (2015)

GluR2–GAPDH complex in multiple sclerosis (MS). (A, B) GluR2–GAPDH complex formation is significantly increased in the plaque of multiple sclerosis (MS). Postmortem samples from control, MS plaque (plaque+) area, and MS nonplaque (plaque−) area were incubated with GluR2 antibody and the precipitated proteins were immunoblotted with GAPDH antibody or GluR2 antibody. The intensity of each protein band for GAPDH (A), GluR2 (B) from all three groups was quantified by densitometry (AIS software, Imaging Research Inc.). Results for each sample are presented as the percentage of the mean of the control samples on the same blot. (*P < 0.05, n = 8, one-way ANOVA). (C–E) GluR2–GAPDH complex formation is significantly increased in experimental autoimmune encephalitis (EAE) mice compared to sham mice. (A) Representative image of western blot analysis of GAPDH (top) and GluR2 (bottom) levels precipitated by GluR2 antibody in extract prepared from mouse spinal cord tissue were incubated with GluR2 antibody. Precipitated proteins were subject to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotted with GAPDH or GluR2 antibody. (D, E) Densitometric analysis of the level of GAPDH and GluR2. The intensity of GAPDH and GluR2 was quantified by densitometry (software: Image J, NIH). Data were analyzed by t-test. (**P < 0.01, n = 3). (F) Clinical EAE scores (mean ± SEM) over time of four groups vaccinated with MOG35-55 on day 0 and treated intraperitoneally daily with TAT-G-Gpep and TAT-G-Gpep-Sc from day 10 (arrow). Starting from day 12, P < 0.05, data were analyzed by Mann–Whitney U test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4402084&req=5

fig01: GluR2–GAPDH complex in multiple sclerosis (MS). (A, B) GluR2–GAPDH complex formation is significantly increased in the plaque of multiple sclerosis (MS). Postmortem samples from control, MS plaque (plaque+) area, and MS nonplaque (plaque−) area were incubated with GluR2 antibody and the precipitated proteins were immunoblotted with GAPDH antibody or GluR2 antibody. The intensity of each protein band for GAPDH (A), GluR2 (B) from all three groups was quantified by densitometry (AIS software, Imaging Research Inc.). Results for each sample are presented as the percentage of the mean of the control samples on the same blot. (*P < 0.05, n = 8, one-way ANOVA). (C–E) GluR2–GAPDH complex formation is significantly increased in experimental autoimmune encephalitis (EAE) mice compared to sham mice. (A) Representative image of western blot analysis of GAPDH (top) and GluR2 (bottom) levels precipitated by GluR2 antibody in extract prepared from mouse spinal cord tissue were incubated with GluR2 antibody. Precipitated proteins were subject to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotted with GAPDH or GluR2 antibody. (D, E) Densitometric analysis of the level of GAPDH and GluR2. The intensity of GAPDH and GluR2 was quantified by densitometry (software: Image J, NIH). Data were analyzed by t-test. (**P < 0.01, n = 3). (F) Clinical EAE scores (mean ± SEM) over time of four groups vaccinated with MOG35-55 on day 0 and treated intraperitoneally daily with TAT-G-Gpep and TAT-G-Gpep-Sc from day 10 (arrow). Starting from day 12, P < 0.05, data were analyzed by Mann–Whitney U test.
Mentions: As shown in Figure1A, co-immunoprecipitation of GAPDH by the GluR2 antibody was significantly greater in protein extracted from MS spinal cord plaques compared to both healthy regions spinal cord from MS patients and healthy control subjects (n = 8, P < 0.05). In contrast, neither the levels of directly immunoprecipitated GluR2 (Fig.1B) nor the expression of GluR2 and GAPDH was significantly different between all groups (Fig. S1A and B). These data suggest an increased level of GluR2–GAPDH complex in MS plaques but not healthy tissue.

Bottom Line: We found that protein complex composed of the GluR2 subunit of AMPA receptors and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was present at significantly higher levels in postmortem tissue from MS patients and in EAE mice, an animal model for MS.This peptide greatly improves neurological function in EAE mice, reduces neuron death, rescues demyelination, increases oligodendrocyte survival, and reduces axonal damage in the spinal cords of EAE mice.More importantly, our peptide has no direct suppressive effect on naive T-cell responses or basal neurotransmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Centre for Addiction and Mental Health Toronto, Ontario, Canada, M5T 1R8.

ABSTRACT

Objective: Multiple sclerosis (MS) is the most common disabling neurological disease of young adults. The pathophysiological mechanism of MS remains largely unknown and no cure is available. Current clinical treatments for MS modulate the immune system, with the rationale that autoimmunity is at the core of MS pathophysiology.

Methods: Experimental autoimmune encephalitis (EAE) was induced in mice with MOG35-55 and clinical scoring was performed to monitor signs of paralysis. EAE mice were injected intraperitoneally with TAT-fusion peptides daily from day 10 until day 30 after immunization, and their effects were measured at day 17 or day 30.

Results: We report a novel target for the development of MS therapy, which aimed at blocking glutamate-mediated neurotoxicity through targeting the interaction between the AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid) receptor and an interacting protein. We found that protein complex composed of the GluR2 subunit of AMPA receptors and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was present at significantly higher levels in postmortem tissue from MS patients and in EAE mice, an animal model for MS. Next, we developed a peptide that specifically disrupts the GluR2 -GAPDH complex. This peptide greatly improves neurological function in EAE mice, reduces neuron death, rescues demyelination, increases oligodendrocyte survival, and reduces axonal damage in the spinal cords of EAE mice. More importantly, our peptide has no direct suppressive effect on naive T-cell responses or basal neurotransmission.

Interpretation: The GluR2 -GAPDH complex represents a novel therapeutic target for the development of medications for MS that work through a different mechanism than existing treatments.

No MeSH data available.


Related in: MedlinePlus