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Cadmium Impairs Autophagy Leading to Apoptosis by Ca2+-Dependent Activation of JNK Signaling Pathway in Neuronal Cells

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Abstract

Autophagy, a process for self-degradation of intracellular components and dysfunctional organelles, is closely related with neurodegenerative diseases. It has been shown that cadmium (Cd) induces neurotoxicity partly by impairing autophagy. However, the underlying mechanism is not fully elucidated. In this study, we show that Cd induced expansion of autophagosomes with a concomitant abnormal expression of autophagy-related (Atg) proteins in PC12 cells and primary murine neurons. 3-MA, a classical inhibitor of autophagy, attenuated Cd-induced expansion of autophagosomes and apoptosis in the cells. Further investigation demonstrated that Cd activated JNK pathway contributing to autophagosome expansion-dependent neuronal apoptosis. This is supported by the findings that pharmacological inhibition of JNK with SP600125 or expression of dominant negative c-Jun markedly attenuated Cd-induced expansion of autophagosomes and abnormal expression of Atg proteins, as well as apoptosis in PC12 cells and/or primary neurons. Furthermore, we noticed that chelating intracellular free Ca2+ ([Ca2+]i) with BAPTA/AM profoundly blocked Cd-elicited activation of JNK pathway and consequential expansion of autophagosomes, abnormal expression of Atg proteins, and apoptosis in the neuronal cells. Similar events were also seen following prevention of [Ca2+]i elevation with EGTA or 2-APB, implying a Ca2+-dependent mechanism involved. Taken together, the results indicate that Cd impairs autophagy leading to apoptosis by Ca2+-dependent activation of JNK signaling pathway in neuronal cells. Our findings highlight that manipulation of intracellular Ca2+ level and/or JNK activity to ameliorate autophagy may be a promising intervention against Cd-induced neurotoxicity and neurodegeneration.

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Abbreviations

AD:

Alzheimer disease

ALS:

Amyotrophic lateral sclerosis

AMPK:

AMP-activated protein kinase

2-APB:

2-Aminoethoxydiphenyl borane

Atg:

Autophagy-related

BAPTA/AM:

1,2-Bis (o-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid tetra (acetoxymethyl) ester

Ca2 + :

Calcium ion

Cd:

Cadmium

CRAC:

Ca2+-release activated Ca2+

DAPI:

4′, 6-Diamidino-2-phenylindole

DMEM:

Dulbecco’s Modified Eagle’s Medium

EGTA:

Ethylene glycol tetra-acetic acid

ER:

Endoplasmic reticulum

Erk1/2:

Extracellular signal-regulated kinase ½

FBS:

Fetal bovine serum

GFP:

Green fluorescent protein

HD:

Huntington's disease

JNK:

C-Jun N-terminal kinase

LacZ:

β-Galacftosidase

LC3:

Microtuble-associated protein 1 light chain 3

3-MA:

3-Methyladenine

MAPK:

Mitogen-activated protein kinase

MDC:

Monodansylcadaverine

mTOR:

Mammalian target of rapamycin

mTORC1:

MTOR complexes 1

PBS:

Phosphate buffered saline

OD:

Optical density

PD:

Parkinson disease

PDL:

Poly-D-lysine

PI3K:

Phosphatidylinositol 3’-kinase

ROS:

Reactive oxygen species

TUNEL:

The terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling

ULK1:

Unc51-like kinase 1

zVAD-fmk:

Z-Val-Ala-Asp-CH2F

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Acknowledgements

This work was supported in part by the grants from National Natural Science Foundation of China (No. 81873781; LC), NIH (CA115414; SH), Project for the Priority Academic Program Development of Jiangsu Higher Education Institutions of China (PAPD-14KJB180010; LC), Natural Science Foundation of Jiangsu Province (BK20180562; CX), and American Cancer Society (RSG-08-135-01-CNE; SH).

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Author notes

  1. Chong Xu and Sujuan Chen have contributed equally to this work.

Authors and Affiliations

  1. Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Chixia District, Nanjing, 210023, People’s Republic of China

    Chong Xu, Sujuan Chen, Ming Xu, Xiaoling Chen, Xiaoxue Wang, Hai Zhang, Xiaoqing Dong, Ruijie Zhang, Xin Chen, Wei Gao & Long Chen

  2. School of Basic Medical Sciences and Clinical Pharmacy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China

    Chong Xu

  3. Synthetic Biology Engineering Lab of Henan Province, School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003, People’s Republic of China

    Sujuan Chen

  4. Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA, 71130-3932, USA

    Shile Huang

  5. Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932, USA

    Shile Huang

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  1. Chong Xu
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Contributions

LC and SH conceived the project. CX, SC and LC designed the experiments. CX, SC and XW performed the experiments. XC, SC, XW, SH and LC analyzed the data. XC, MX, HZ, XD, RZ, XC and WG contributed reagents/materials/analysis tools. CX, SH and LC wrote the paper.

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Correspondence to Shile Huang or Long Chen.

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Xu, C., Chen, S., Xu, M. et al. Cadmium Impairs Autophagy Leading to Apoptosis by Ca2+-Dependent Activation of JNK Signaling Pathway in Neuronal Cells. Neurochem Res 46, 2033–2045 (2021). https://doi.org/10.1007/s11064-021-03341-x

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