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Low Levels of Prohibitin in Substantia Nigra Makes Dopaminergic Neurons Vulnerable in Parkinson’s Disease

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Abstract

Since substantia nigra (SN) and ventral tegmental area (VTA) dopaminergic neurons are, respectively, susceptible or largely unaffected in Parkinson’s disease (PD), we searched for protein(s) that regulates this differential sensitivity. Differentially, expressed proteins in SN and VTA were investigated employing two-directional gel electrophoresis- matrix-assisted laser desorption ionization time of flight (MALDI-TOF-TOF) analyses. Prohibitin, which is involved in mitochondrial integrity, was validated using immunoblot, qRT-PCR, and immunohistochemistry in normal mice as well as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-model, PD postmortem human brains, and PD cybrids. In prohibitin over-expression, differentiated SH-SY5Y neurons were investigated for their susceptibility to PD neurotoxin, 1-methyl-4-phenyl-pyridnium (MPP+). Prohibitin, Hsc73, and Cu-Zn superoxide dismutase (Cu-Zn SOD) were highly expressed in VTA, whereas heat shock protein A8 (HSPA8) and 14-3-3ζ/δ were 2-fold more in SN. Prohibitin level was transiently increased in SN but unaltered in VTA on the third day of MPTP-induced mice, whereas in PD human brains, prohibitin was depleted in both these regions. Parallel to mouse SN, an enhanced prohibitin expression was found in human PD cybrids. In MPP+-induced cellular model of PD, reduction in prohibitin level was found to be associated with a loss in its binding with Ndufs3, a mitochondrial complex I protein partner. Prohibitin over-expression resisted MPP+-induced neuronal death by restoring mitochondrial membrane potential, preventing reactive oxygen species generation and cytochrome c release into cytosol. These protective phenomena exerted by prohibitin over-expression altogether hinder caspase 3 activation induced by MPP+. These results imply that prohibitin is an important negotiator protein that regulates dopaminergic cell death in SN and their protection in VTA in PD.

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Acknowledgements

D.D., N.A., R.S., and P.R.K. are research fellows of the Council of Scientific and Industrial Research (CSIR), Govt. of India. E.B. and A.N. received fellowships from Department of Biotechnology, Govt. of India. The study was initiated as a major laboratory program (MLP) of CSIR-IICB and later on financed by the network project of the 12th Five-Year Plan of CSIR, “Neurodegenerative Diseases: Causes and Corrections” (miND: BSC 0115). Continuation of the program was funded by Department of Higher Education, Government of Kerala, through Mahatma Gandhi University, Kottayam. We thank Prof. S.K. Shankar, M.D. and Dr. Anita Mahadevan, M.D., the principal and associate coordinators, respectively, of Human Brain Bank at NIMHANS, Bangalore for the midbrain postmortem human brain samples. We also thank Dr. Arianne L. Theiss, Baylor Research Institute, TX, USA for providing pEGFPN1-PHB vector construct. A special thanks to Mr. Sandip Chakraborty and Mr. Diptadeep Sarkar are due for their technical support in MALDI-TOF-TOF analysis and confocal microscopy, respectively.

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  1. Kochupurackal P Mohanakumar

    Present address: Inter University Centre for Biomedical Research and Super Speciality Hospital, Mahatma Gandhi University Campus at Thalappady, Rubber Board P.O, Kottayam, Kerala, 686009, India

Authors and Affiliations

  1. Laboratory of Clinical and Experimental Neuroscience, Division of Cell Biology and Physiology, CSIR—Indian Institute of Chemical Biology, 4-Raja S. C. Mullick Road, Jadavpur, Kolkata, 700 032, India

    Debashis Dutta, Nilufar Ali, Emili Banerjee, Raghavendra Singh, Amit Naskar, Ramesh Kumar Paidi & Kochupurackal P Mohanakumar

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The experimental protocol met the National Guidelines (CPCSEA, Ministry of Environment, Forests and Climate Changes, Govt. of India) on the Proper Care and Use of Animals in Laboratory Research and was approved by the Animal Ethics Committee of the Institute.

The study protocol was approved by the institutional Human Ethics Committee strictly following the guidelines of Indian Council of Medical Research, Ministry of Health and Family Welfare, Govt. of India.

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Electronic supplementary material

Supplementary Fig. 1

Categorization of proteins identified from 2-DE gel using iProclass database of Protein Information Resource (PIR, www.pir.georgetown.edu) gene ontology database according to Cellular component, Molecular function and Biological process. In each pie chart the total number of proteins present in each category is given in parenthesis. (GIF 63 kb)

High resolution image (TIFF 5220 kb)

Supplementary Fig. 2

Protein interactome analysis using String; v 9.0 showed PHB to interact with several mitochondrial complex I subunits, some of which showed direct interactions are Ndufs3, Ndufv2, and Ndufv1. The interactions were based on neighborhood, gene fusion, text-mining, co-occurrence, co-expression and database, and was with a with high confidence level of 0.7. (GIF 17 kb)

High resolution image (TIFF 1568 kb)

Supplementary Fig. 3

Immunostaining by Alexa Fluor 488 tagged secondary antibodies (green color) against tyrosine hydroxylase (TH) present inside the dopamine (DA) neurons of substantia nigra pars compacta (SN) and ventral tegmental area (VTA) of different groups of mice (control, MPTP 3d – animals analyzed after 3 days of last MPTP injection, and MPTP 7d - animals analyzed after 7 days of last MPTP injection). SN and VTA are marked in the control image. Scale bar 100 μm. (GIF 11 kb)

High resolution image (TIFF 761 kb)

Supplementary Fig. 4

(A) Human embryonic kidney-293T (HEK-293T) cells were transduced with adenoviruses coding human PHB gene tagged with emerald GFP and kept for 24 h. Fluorescent images show enhanced level of PHB-GFP expression with increasing volume of adenovirus stock volume given to the cells (10, 20 and 40 μl). Scale bar was 200 μm. (B) Immunoblot shows enhanced level of PHB-GFP (60 KD) along with indigenous PHB (32 KD) in virus-treated and non-treated cells (NT). PHB-GFP is absent in NT sample. β-actin was used as the loading control (44 KD). (GIF 11 kb)

High resolution image (TIFF 1031 kb)

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Dutta, D., Ali, N., Banerjee, E. et al. Low Levels of Prohibitin in Substantia Nigra Makes Dopaminergic Neurons Vulnerable in Parkinson’s Disease. Mol Neurobiol 55, 804–821 (2018). https://doi.org/10.1007/s12035-016-0328-y

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