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Selective removal of misfolded SOD1 delays disease onset in a mouse model of amyotrophic lateral sclerosis

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Abstract

Background

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. There is no cure currently. The discovery that mutations in the gene SOD1 are a cause of ALS marks a breakthrough in the search for effective treatments for ALS. SOD1 is an antioxidant that is highly expressed in motor neurons. Human SOD1 is prone to aberrant modifications. Familial ALS-linked SOD1 variants are particularly susceptible to aberrant modifications. Once modified, SOD1 undergoes conformational changes and becomes misfolded. This study aims to determine the effect of selective removal of misfolded SOD1 on the pathogenesis of ALS.

Methods

Based on the chaperone-mediated protein degradation pathway, we designed a fusion peptide named CT4 and tested its efficiency in knocking down intracellularly misfolded SOD1 and its efficacy in modifying the pathogenesis of ALS.

Results

Expression of the plasmid carrying the CT4 sequence in human HEK cells resulted in robust removal of misfolded SOD1 induced by serum deprivation. Co-transfection of the CT4 and the G93A-hSOD1 plasmids at various ratios demonstrated a dose-dependent knockdown efficiency on G93A-hSOD1, which could be further increased when misfolding of SOD1 was enhanced by serum deprivation. Application of the full-length CT4 peptide to primary cultures of neurons expressing the G93A variant of human SOD1 revealed a time course of the degradation of misfolded SOD1; misfolded SOD1 started to decrease by 2 h after the application of CT4 and disappeared by 7 h. Intravenous administration of the CT4 peptide at 10 mg/kg to the G93A-hSOD1 reduced human SOD1 in spinal cord tissue by 68% in 24 h and 54% in 48 h in presymptomatic ALS mice. Intraperitoneal administration of the CT4 peptide starting from 60 days of age significantly delayed the onset of ALS and prolonged the lifespan of the G93A-hSOD1 mice.

Conclusions

The CT4 peptide directs the degradation of misfolded SOD1 in high efficiency and specificity. Selective removal of misfolded SOD1 significantly delays the onset of ALS, demonstrating that misfolded SOD1 is the toxic form of SOD1 that causes motor neuron death. The study proves that selective removal of misfolded SOD1 is a promising treatment for ALS.

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Availability of supporting data

All data published in the paper will be available upon request.

Abbreviations

ALS:

Amyotrophic lateral sclerosis

CMA:

Chaperone-mediated autophagy

CTM:

Chaperone-mediated autophagy targeting motif

DBR:

Derlin-1 binding region

Derlin-1:

Degradation in endoplasmic reticulum protein 1

FALS:

Familial amyotrophic lateral sclerosis

NSC:

Neural stem cell

WT:

Wild-type

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Acknowledgements

The authors would like to acknowledge the assistance of Dr Lynda Kong in editing the manuscript.

Funding

The study was supported by Grants from ALS Society of Canada, Brain Canada and By-Health China.

Author information

Author notes

  1. Teng Guan, Ting Zhou and Xiaosha Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Human Anatomy and Cell Science, Max Rady College of Medicine, University of Manitoba, Winnipeg, MB, Canada

    Teng Guan, Ting Zhou, Xiaosha Zhang, Ying Guo, Chaoxian Yang, Justin Lin, Jiasi Vicky Zhang, Yongquan Cheng, Hassan Marzban & Jiming Kong

  2. Department of Pharmacy, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China

    Ting Zhou

  3. Department of Forensic Medicine, Hebei North University, Zhangjiakou, China

    Ying Guo

  4. Department of Neurobiology, Southwest Medical University, Luzhou, China

    Chaoxian Yang

  5. Brain Research Centre and Department of Medicine, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, BC, Canada

    Yu Tian Wang

  6. Department of Human Anatomy and Cell Science, Max Rady College of Medicine, University of Manitoba, 745 Bannatyne Avenue, Winnipeg, MB, R3E 0J9, Canada

    Jiming Kong

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  1. Teng Guan
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Contributions

TG coordinated the project and drafted the manuscript. TZ performed most of the in vivo experiments. XZ conducted plasmid construction and in vitro validation. YG, CY, YC aided in animal experiments. JL, JVZ helped cell-based experiments. HM, YTW made a conceptual contribution and reviewed the manuscript. JK initiated and supervised the project. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jiming Kong.

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Supplementary Information

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18_2023_4956_MOESM1_ESM.tiff

Supplementary file1 Supplementary Figure 1. Knockdown of misfolded SOD1 by CT4 in glial cells. (A) G93A-hSOD1 expressing astrocytes (GFAP+), oligodendrocyte precursor cells (OPCs, PDGFRα+), and mature oligodendrocytes (MBP+) were treated with 5 µM CT4 for 2 hours. Cells treated with mCT4 were used as controls. Scale bar = 20 μm. G93A-hSOD1 fluorescence intensities were measured in each type of cell. n = 6 from 3 separate cultures. (B) Knockdown of misfolded SOD1 in G93A-hSOD1 mice by intravenous injection of CT4 or mCT4 at 10 mg/kg daily for 3 days. Immunostaining images with anti-misfolded SOD1 (B8H10) antibody and glial markers in the lumbar spinal cord are shown. Scale bar: 100 μm (for images in lower magnification) and 20 μm (higher magnification). G93A-hSOD1 fluorescence intensities (mean ± SD) were measured in each cell type and analyzed with two-way ANOVA followed by Sidak’s multiple comparisons test. **P<0.01 (TIFF 10628 KB)

18_2023_4956_MOESM2_ESM.tiff

Supplementary file2 Supplementary Figure 2. Short-term knockdown of misfolded SOD1 by CT4 in vivo. (A) Western blotting of misfolded SOD1 with the A5C3 antibody in mice expressing WT-hSOD1 and G93A-hSOD1 at the age of 150 days. (B) The G93A-hSOD1 mice received intravenous injections of either mCT4 or CT4 peptide at 10 mg/kg body weight daily for three days at the age of 100 days. Spinal cords were harvested 24 hours after the last injection. Equal volumes of lysates with a total of 10 µg protein were applied to 12% TGX Stain-Free polyacrylamide gels. Total protein generated by stain-free visualization was used to ensure equivalent loading. A two-tailed t-test was performed to establish significant differences with the control group. *P < 0.05; **P < 0.01. Shown are mean ± SD. n = 4 in mCT4 group and 3 in CT4 group (TIFF 4100 KB)

18_2023_4956_MOESM3_ESM.tiff

Supplementary file3 Supplementary Figure 3. No toxicity was observed in wild-type mice exposed to CT4. Beginning at 60 days of age, wild-type mice received an IP injection of CT4 peptide at a dose of 10 mg/kg q.o.d until 173 days of age. Sections of mouse brain, heart, spinal cord, kidney, liver, muscle, lung, and spleen were stained with H&E. Scale bar represents 100 μm.  (TIFF 14391 KB)

Supplementary file4 Supplementary Table 1. List of primary antibodies used in experiments. (DOCX 15 KB)

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Guan, T., Zhou, T., Zhang, X. et al. Selective removal of misfolded SOD1 delays disease onset in a mouse model of amyotrophic lateral sclerosis. Cell. Mol. Life Sci. 80, 304 (2023). https://doi.org/10.1007/s00018-023-04956-9

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