Verebne Tar 2016 Abstract MitoFit Science Camp 2016

From Bioblast
Investigating the role of the proteasome activator PA200 in mitochondrial homeostasis in a cellular model for Huntington’s disease.


Krisztina Tar

Czinege R, Yazaki R, Virag L, Tar K (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

Huntington’s disease (HD) is a progressive, incurable, autosomal dominant hereditary disease that affects significant number of patients. The disease belongs to the neurodegenerative disorders where intraneuronal inclusions are hallmark of the disease. The cause of HD is the expansion of polyglutamine (polyQ) repeat in exon 1 of huntingtin protein (Htt) that makes it prone to misfold and aggregate forming inclusion bodies, moreover, polyQ length correlates disease onset and severity.

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate peptide and protein turnover in vitro. We provided evidence for an additional regulatory function of the proteasome in mitochondrial homeostasis [1]. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. We demonstrated that the mitochondrial fission protein Dnm1 is degraded by the proteasome and that proteasome-mediated Dnm1 turnover involves the proteasome activator Blm10. We have also shown that loss of BLM10/PA200 upon expression of mutant huntingtin protein (mHtt) leads to a severe growth defect, increased aggregate formation of mHtt and excessive mitochondrial fragmentation. Assuming that the impact of Blm10 on Dnm1 turnover is conserved in mammals, we propose that upregulation of its mammalian ortholog PA200 might provide a neuroprotective function through the regulated degradation of Drp1 and is involved in the altered mitochondrial dynamics and function during development of HD.

We generated PA200 knock-down SH SY5Y human neuroblastoma cell lines (shPA200) using shRNA lentiviral technology. We looked at Drp1 turnover by cycloheximide (CHX) chase assay and have shown that PA200 is required for the correct degradation of Drp1 by the proteasome. Furthermore, we have observed that loss of PA200 leads to dramatic alteration of mitochondrial morphology. We checked mitochondrial metabolism by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in shPA200 and control cells using Seahorse XF Analyzer. Our results show that loss of PA200 leads to impaired fatty acid oxidation and glycolysis.

β€’ O2k-Network Lab: HU Debrecen Virag L

Labels: MiParea: Respiration, nDNA;cell genetics, Genetic knockout;overexpression, mt-Medicine  Pathology: Neurodegenerative  Stress:Oxidative stress;RONS  Organism: Human  Tissue;cell: Nervous system, Other cell lines 

Event: B3  MitoFit Science Camp 2016 


Dept Med Chem, Fac Med, Univ Debrecen, Hungary. - [email protected]

Abstract continued

Moreover, Drp1 is stabilized in shPA200 cells upon expressing different length of mHtt while in its respective control Drp1 level is dramatically decreased. A previous study showed that mutant Htt binds to Drp1, changes its structure and increases its enzymatic activity [2]. The mechanistic details are unknown. Presumably in mammals the Drp1 turnover is mediated by PA200-proteasomes, is required for normal mitochondrial function and provides a cytoprotective function under conditions that induce mitochondrial stress such as expression of mHtt, aggregate formation and disease progression.

Furthermore, the characterisation of PA200-proteasomes, mitochondria and mHtt interplay would provide us with more data about their physiological role and pathological effect on neurodegenerative diseases and that knowledge and experience will help to better understand and influence diseases.


  1. Tar K, Dange T, Yang C, Yao Y, Bulteau AL, Salcedo EF, Braigen S, Bouillaud F, Finley D (2014) Proteasomes associated with the Blm10 activator protein antagonize mitochondrial fission through degradation of the fission protein Dnm1. J Biol Chem 289:12145-56.
  2. Song W, Chen J, Petrilli A, Liot G, Klinglmayr E, Zhou Y, Poquiz P, Tjong J, Pouladi MA, Hayden MR, Masliah E, Ellisman M, Rouiller I, Schwarzenbacher R, Bossy B, Perkins G, Bossy-Wetzel E (2011) Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity. Nat Med 17:377-82.
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