Taking Out the Mitochondria in Parkinson’s Disease

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Parkinson’s disease (PD) is a neurodegenerative disease marked by loss of dopaminergic neurons in the substantia nigra. Mutations in the gene that encodes a ubiquitin ligase PARK2/Parkin are known to cause autosomal recessive forms of familial PD¹. Parkin plays a key role in mitochondrial homeostasis by regulating a specialized form of autophagy called mitophagy, the clearance of defective or damaged mitochondria by lysosomes².

How does altered mitochondrial homeostasis contribute to PD?

Currently, this is not well understood. One could imagine cell-autonomous effects, factors that might directly alter the viability of dopaminergic neurons, or non-cell autonomous contributions³. For example, accumulation of α-synuclein (α-syn) is one of the hallmarks of PD. Cell-autonomous mechanisms of α-syn clearance have been reported in neurons. However, non-neuronal cells like astrocytes and microglia are likely to contribute to neuronal toxicity via non-cell autonomous effects by indirectly contributing to α-syn accumulation by internalizing and degrading extracellular α-syn.

Might altered microglia be particularly sensitive to Parkin-dependent maintenance of mitochondrial homeostasis?

At the recent Alzheimer’s Disease/Parkinson’s Disease (ADPD) meeting, a group from the Brain and Spine Institute in Paris, France reported that altered Parkin-dependent mitophagy may be linked to inflammasome activation in microglia⁴. Microglia are brain-resident macrophages that have broad functions in both the developing and mature brain⁵. Increased microglia-mediated neuroinflammation has been reported in PD patients⁶. Inflammation, characterized by the release of proinflammatory cytokines, can be initiated by the mobilization of the NLRP3 inflammasome in microglia⁷. Using macrophages from PARK2 patients and from mice lacking PARK2 as a model for microglia, the authors observed enhanced NLRP3 inflammasome formation, suggesting that the NLRP3 inflammasome could be regulated by PARK2.

How might Parkin, a protein that ubiquitinates and marks proteins for degradation, regulate the NLRP3 inflammasome pathway?

The authors provided evidence that Parkin regulates inflammasome activation via two mechanisms—mitophagy-dependent regulation of the inflammasome complex and transcriptional regulation of inhibitory proteins upstream of the inflammasome expression pathway. In the latter case, the authors proposed that Parkin could regulate A20, a negative regulator of the NF-κB pathway that is uniquely expressed in microglia in the brain. Thus, in the absence of normal Parkin function, A20 inhibition is tempered resulting in NF-κB inflammasome overactivation.

Several pieces of this puzzle remain, however. For example, Parkin normally marks proteins for degradation by ubiquitinating specific proteins. A20, however, is an inhibitor of the NF-κB pathway, suggesting that Parkin might regulate an unknown protein upstream of A20⁸. As A20 is a microglia-enriched protein, alterations in this pathway may contribute to the progression of inflammation in the context of PARK2 mutations.

How might one identify Parkin-ubiquitinated proteins upstream of A20 to more fully characterize this pathway?

One way to do it is to use proteomics.

This is the same problem that investigators at Genentech faced when they sought to investigate molecular pathways that might mediate Parkin/USP30-depedent mitophagy⁹. This is a difficult problem because ubiquitinated substrates are difficult to predict. Using proteomics technology provides a more high-throughput and unbiased approach. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a powerful technology that can identify and quantify thousands of peptides in a complex mixture including cells, tissues, and bodily fluids. By LC-MS/MS, not only can peptide sequences be determined, modifications on amino acid side chains can also be identified by incorporating the modification mass into protein database searching.

However, low-abundant peptides carrying targeted post-translational modifications can be a challenge to identify, an issue the Genentech authors faced. To overcome this issue, they used a ubiquitin remnant motif antibody pulldown technique to enrich ubiquitinated substrates. Using this method, they were able to detect thousands of ubiquitinated peptides in parkin-overexpressing and USP-knock-down HEK293 cell lines. Their screen was quite successful in that 12 novel mitochondrial proteins, oppositely ubiquitinated by Parkin and USP30, were discovered. Of the 12, two proteins, TOM20 and MIRO1, were selected for verification because their ubiquitination levels increased significantly with Parkin overexpression and USP30 knockdown. Additional experiments confirmed that deubiquitination of TOM20 mediated by USP30 plays an important role in inhibiting mitophagy mediated by Parkin, representing a novel signaling pathway that regulates mitophagy.

Why It Matters...

Both neuroinflammation and mitochondrial dysfunction are involved in PD pathogenesis. Identification of ubiquitinated substrates using LC-MS/MS, coupled with immuno-affinity enrichment of modified substrates, revealed ubiquitinated substrates and key molecular mechanisms that drive mitophagy. This strategy may also be valuable to identify Parkin substrate upstream of A20 in neuroinflammation models, potentially revealing a new therapeutic target to combat neurodegeneration.  

Research more about inflammasome signaling.

References:

1. Kitada T., et al., Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. 1998 Nature 392 (6676): 605-8
2. Nguyen M., et al. Synaptic, Mitochondrial, and Lysosomal Dysfunction in Parkinson's Disease. 2019 Trends Neurosci 42(2): 140-149
3. Filippini A., et al. α-Synuclein and Glia in Parkinson’s Disease: A Beneficial or a Detrimental Duet for the Endo-Lysosomal System? 2019 Cellular and Molecular Neurobiology 39 (2): 161-168
4. Mouston-Liger F., et al. NLRP3 Inflammasome Overactivation in PARK2-linked Parkinson’s Disease. 2019 Alzheimer’s Disease/Parkinson’s Disease Biannual Meeting Symposium 44
5. Hammond T.R., et al., Microglia and the Brain: Complementary Partners in Development and Disease. 2018 Annual Review Cell and Development Biology 34: 523-544
6. Mouton-Liger F., et al. PINK1/Parkin-Dependent Mitochondrial Surveillance: From Pleiotropy to Parkinson's Disease. 2017 Frontiers in Molecular Neuroscience 10: 120
7. He Y., et al., Mechanism and Regulation of NLRP3 Inflammasome Activation. 2016 Trends in Biochemical Sciences 41(12): 1012-1021 
8. Li Z., et al. A20 as a novel target for the anti-neuroinflammatory effect of chrysin via inhibition of NF-κB signaling pathway.2019 Brain, Behavior, and Immunity 79:288-235
9. Bingol B., et al. The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy 2014 Nature 510(7505):370-5