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The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis

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Abstract

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), β-tubulin and β-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.

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Abbreviations

AMC:

7-Aminomethyl-4-coumarin

DMSO:

Dimethyl sulfoxide

DNP:

Dinitrophenyl

dpi:

Days post-immunization

EAE:

Experimental autoimmune encephalomyelitis

ECL:

Enhanced chemioluminescence

HRP:

Horseradish peroxidase

i.t.:

Intrathecal

MBP:

Myelin basic protein

MS:

Multiple sclerosis

NFH:

Neurofilament heavy (200 kDa) protein

NFL:

Neurofilament light (69 kDa) protein

NFM:

Neurofilament medium (150 kDa) protein

NFPs:

Neurofilament proteins

PAGE:

Polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene difluoride

SDS:

Sodium dodecyl sulfate

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Acknowledgements

The authors wish to thank Dr. Erin Milligan from the Department of Neurosciences at UNM-SOM for helping with the i.t. injections. This work was supported by PHHS Grant NS057755 from the National Institutes of Health.

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Authors and Affiliations

  1. Department of Cell Biology and Physiology, University of New Mexico - Health Sciences Center, Albuquerque, NM, USA

    Suzanne M. Smerjac, Jianzheng Zheng, Che-Lin Hu & Oscar A. Bizzozero

  2. Department of Cell Biology and Physiology, University of New Mexico School of Medicine, 1 University Of New Mexico, MSC08 4750, Albuquerque, NM, 87131, USA

    Oscar A. Bizzozero

Authors
  1. Suzanne M. Smerjac
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  2. Jianzheng Zheng
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  3. Che-Lin Hu
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  4. Oscar A. Bizzozero
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Correspondence to Oscar A. Bizzozero.

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Smerjac, S.M., Zheng, J., Hu, CL. et al. The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis. Neurochem Res 43, 2277–2287 (2018). https://doi.org/10.1007/s11064-018-2648-y

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