David N. Hauser

p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both

Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including Parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin’s translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3-/- mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin substrates other than VDAC1.

Mitochondrial dysfunction and oxidative stress in Parkinson's disease and monogenic parkinsonism

The pathogenic mechanisms that underlie Parkinsons disease remain unknown. Here, we review evidence from both sporadic and genetic forms of Parkinsons disease that implicate both mitochondria and oxidative stress as central players in disease pathogenesis. A systemic deficiency in complex I of the mitochondrial electron transport chain is evident in many patients with the disease. Oxidative stress caused by reactive metabolites of dopamine and alterations in the levels of iron and glutathione in the substantia nigra accompany this mitochondrial dysfunction. Recent evidence from studies on the genetic forms of parkinsonism with particular stress on DJ-1, parkin, and PINK-1 also suggest the involvement of mitochondria and oxidative stress.

The Analytic Hierarchy Process in an uncertain environment: A simulation approach

Abstract Traditionally, decision makers were forced to converge ambiguous judgments to a single point estimate in order to describe a pairwise relationship between alternatives relative to some criterion for use in the Analytic Hierarchy Process (AHP). Since many circumstances exist which make such a convergence difficult, confidence in the outcome of an ensuing AHP synthesis may be reduced. Likewise, when a group of decision makers cannot arrive at a consensus regarding a judgment, some members of the group may simply lose confidence in the overall synthesis if they lack faith in some of the judgments. The AHP utilizes point estimates in order to derive the relative weights of criteria, sub-criteria, and alternatives which govern a decision problem. However, when point estimates are difficult to determine, distributions describing feasible judgments may be more appropriate. Using simulation, we will demonstrate that levels of confidence can be developed, expected weights can be calculated and expected ranks can be determined. It will also be shown that the simulation approach is far more revealing than traditional sensitivity analysis.

mRNA expression, splicing and editing in the embryonic and adult mouse cerebral cortex

The complexity of the adult brain is a result of both developmental processes and experience-dependent circuit formation. One way to look at the differences between embryonic and adult brain is to examine gene expression. Previous studies have used microarrays to address this in a global manner. However, the transcriptome is more complex than gene expression levels alone, as alternative splicing and RNA editing generate a diverse set of mature transcripts. Here we report a high-resolution transcriptome data set of mouse cerebral cortex at embryonic and adult stages using RNA sequencing (RNA-Seq). We found many differences in gene expression, splicing and RNA editing between embryonic and adult cerebral cortex. Each data set was validated technically and biologically, and in each case we found our RNA-Seq observations to have predictive validity. We provide this data set and analysis as a resource for understanding gene expression in the embryonic and adult cerebral cortex.

The G2385R variant of leucine-rich repeat kinase 2 associated with Parkinson's disease is a partial loss-of-function mutation

Autosomal-dominant missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a common genetic cause of PD (Parkinsons disease). LRRK2 is a multidomain protein with kinase and GTPase activities. Dominant mutations are found in the domains that have these two enzyme activities, including the common G2019S mutation that increases kinase activity 2-3-fold. However, there is also a genetic variant in some populations, G2385R, that lies in a C-terminal WD40 domain of LRRK2 and acts as a risk factor for PD. In the present study we show that the G2385R mutation causes a partial loss of the kinase function of LRRK2 and deletion of the C-terminus completely abolishes kinase activity. This effect is strong enough to overcome the kinase-activating effects of the G2019S mutation in the kinase domain. Hsp90 (heat-shock protein of 90 kDa) has an increased affinity for the G2385R variant compared with WT (wild-type) LRRK2, and inhibition of the chaperone binding combined with proteasome inhibition leads to association of mutant LRRK2 with high molecular mass native fractions that probably represent proteasome degradation pathways. The loss-of-function of G2385R correlates with several cellular phenotypes that have been proposed to be kinase-dependent. These results suggest that the C-terminus of LRRK2 plays an important role in maintaining enzymatic function of the protein and that G2385R may be associated with PD in a way that is different from kinase-activating mutations. These results may be important in understanding the differing mechanism(s) by which mutations in LRRK2 act and may also have implications for therapeutic strategies for PD.

Dopamine quinone modifies and decreases the abundance of the mitochondrial selenoprotein glutathione peroxidase 4.

Oxidative stress and mitochondrial dysfunction are known to contribute to the pathogenesis of Parkinsons disease. Dopaminergic neurons may be more sensitive to these stressors because they contain dopamine (DA), a molecule that oxidizes to the electrophilic dopamine quinone (DAQ) which can covalently bind nucleophilic amino acid residues such as cysteine. The identification of proteins that are sensitive to covalent modification and functional alteration by DAQ is of great interest. We have hypothesized that selenoproteins, which contain a highly nucleophilic selenocysteine residue and often play vital roles in the maintenance of neuronal viability, are likely targets for the DAQ. Here we report the findings of our studies on the effect of DA oxidation and DAQ on the mitochondrial antioxidant selenoprotein glutathione peroxidase 4 (GPx4). Purified GPx4 could be covalently modified by DAQ, and the addition of DAQ to rat testes lysate resulted in dose-dependent decreases in GPx4 activity and monomeric protein levels. Exposing intact rat brain mitochondria to DAQ resulted in similar decreases in GPx4 activity and monomeric protein levels as well as detection of multiple forms of DA-conjugated GPx4 protein. Evidence of both GPx4 degradation and polymerization was observed following DAQ exposure. Finally, we observed a dose-dependent loss of mitochondrial GPx4 in differentiated PC12 cells treated with dopamine. Our findings suggest that a decrease in mitochondrial GPx4 monomer and a functional loss of activity may be a contributing factor to the vulnerability of dopaminergic neurons in Parkinsons disease.

Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution

Background: LRRK2 mutations are causative for Parkinson disease, but regulation of LRRK2 remains elusive. Results: Arsenite induces loss of LRRK2 Ser910/Ser935 phosphorylation and 14-3-3 binding, increased self-association, attenuated kinase activity and GTP binding, and translocation to centrosomes. Conclusion: LRRK2 is regulated by arsenite-induced signaling and oxidative stress. Significance: Understanding LRRK2 regulation will provide novel approaches toward developing therapeutic tools targeting LRRK2 activity. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function.

Astrocytes in Parkinson's disease and DJ-1.

Since the German anatomist Rudolf Virchow first coined the term neuroglia in 1846, our ideas about glial cells have moved from viewing them simply as the glue that holds neurons in place. We now know that glia are intimately involved in the lives of neurons, with functions ranging from nurturing developing synapses to actually facilitating the transmission of neuronal information (Eroglu & Barres 2010). Furthermore, glial cells may play detrimental roles in the pathogenesis of nervous system diseases. As an example of this, work from Hinkle and colleagues in this issue of the Journal of Neurochemistry highlight a potential role of astrocytes in Parkinson’s disease associated with mutations in DJ-1 (Mullett & Hinkle 2011). Parkinson’s disease (PD) has been considered a disease of neuronal origin due to the degeneration of groups of neurons, including dopaminergic cells in the substantia nigra pars compacta (SNpc). Mitochondrial complex I dysfunction, associated with accumulation of reactive oxygen species, has been suggested to be important in the loss of neurons in PD. This hypothesis is supported by multiple lines of evidence including the observations that; (1) post mortem biochemical studies have demonstrated lower complex I activity in the SNpc of PD brains compared to controls (Schapira 2007); (2) inhibition of complex I with rotenone or MPTP reproduces the pathological degeneration of the SNpc in mammalian models (Cannon & Greenamyre 2010); and (3) the protein products of genes known to cause familial forms of parkinsonism have clear roles in the maintenance of functional mitochondria (Thomas et al. 2011). A rare form of autosomal recessive parkinsonism is caused by mutations or deletions of the gene coding for the protein DJ-1 (Bonifati et al. 2003). DJ-1 is known to have a role in oxidative stress responses in cells and this is thought to be important in the preservation of neuronal viability. Some of the mechanisms involved are known. For example, the oxidation of a specific cysteine residue in DJ-1 is required to protect cells against mitochondrial damage (Blackinton et al. 2009). However, previous studies have focused on cell-autonomous effects of DJ-1. The work presented here by the Hinkle group further supports their previous findings which suggest a novel mode of action for DJ-1 in which it protects neurons via an astrocyte-mediated mechanism (Mullett & Hinkle 2009). Using a new method of detecting neuronal death in astrocyte/neuron co-cultures, the authors demonstrate that the down-regulation of astrocytic DJ-1 diminishes their ability to protect neurons from stressors. In particular, they show that neuroprotection is selective for drugs that inhibit mitochondrial complex I. However, the authors also found that the neuronal death in their DJ-1 deficient co-cultures treated with these inhibitors could not be rescued by the addition of antioxidants. They also demonstrate that the mechanism underlying astrocyte-mediated protection is not due to the release of the antioxidant glutathione or an up-regulation of astrocytic heme oxygenase-1. Taken together, these findings suggest that astrocytic DJ-1 is necessary for astrocyte-mediated neuroprotection and that the mechanism underlying this phenomenon is selective for complex I inhibitors and is not mediated by an oxidative stress response. The notion that dysfunctional astrocytes may contribute to the death of dopaminergic neurons in the SNpc has several consequences on how we view the pathogenesis of PD and possible treatments. Predominantly, these results say that even where there is a single gene cause of parkinsonism, we should consider models that involve more than just dopamine neurons. In fact, we should consider astrocytes and other non-neuronal cells as possible contributors to the early pathogenic mechanisms. A smaller, but also important point, is that when considering ways in which to treat PD we may need to consider whether the glial environment is supportive or not; treating only neurons may provide only part of the answer. Although the findings presented in this issue by the Hinkle group convincingly show a role for astrocytic DJ-1 in astrocyte-mediated neuroprotection, there are a number of areas that would merit further investigation. If DJ-1 is fundamentally an oxidative stress response protein, then is the neuroprotection also related to oxidative stress? The data presented by Mullet and Hinkle suggests that the effects are not due to, for example, antioxidant release from astrocytes, which may have been an obvious link. The authors propose that soluble factors are released from astrocytes, but these still need to be identified before more detailed mechanistic studies are undertaken. Furthermore, most of the current experiments use dissociated cultured cells. It is known that astrocytes express DJ-1 in vivo, and so it would seem important to determine whether astrocytic DJ-1 is important for neuroprotection in the intact brain. Overall, the data presented here identifies an exciting possibility, that neurodegeneration in a defined form of genetic parkinsonism may involve non-cell autonomous events. Identifying the underlying mechanisms is an important next step and can be facilitated by the types of models presented by Mullet and Hinkle.

Post-Translational Decrease in Respiratory Chain Proteins in the Polg Mutator Mouse Brain

Mitochondrial DNA damage is thought to be a causal contributor to aging as mice with inactivating mutations in polymerase gamma (Polg) develop a progeroid phenotype. To further understand the molecular mechanisms underlying this phenotype, we used iTRAQ and RNA-Seq to determine differences in protein and mRNA abundance respectively in the brains of one year old Polg mutator mice compared to control animals. We found that mitochondrial respiratory chain proteins are specifically decreased in abundance in the brains of the mutator mice, including several nuclear encoded mitochondrial components. However, we found no evidence that the changes we observed in protein levels were the result of decreases in mRNA expression. These results show that there are post-translational effects associated with mutations in Polg.

Use of cysteine-reactive cross-linkers to probe conformational flexibility of human DJ-1 demonstrates that Glu18 mutations are dimers.

The oxidation of a key cysteine residue (Cys106) in the parkinsonism‐associated protein DJ‐1 regulates its ability to protect against oxidative stress and mitochondrial damage. Cys106 interacts with a neighboring protonated Glu18 residue, stabilizing the Cys106‐SO2− (sulfinic acid) form of DJ‐1. To study this important post‐translational modification, we previously designed several Glu18 mutations (E18N, E18D, E18Q) that alter the oxidative propensity of Cys106. However, recent results suggest these Glu18 mutations cause loss of DJ‐1 dimerization, which would severely compromise the proteins function. The purpose of this study was to conclusively determine the oligomerization state of these mutants using X‐ray crystallography, NMR spectroscopy, thermal stability analysis, circular dichroism spectroscopy, sedimentation equilibrium ultracentrifugation, and cross‐linking. We found that all of the Glu18 DJ‐1 mutants were dimeric. Thiol cross‐linking indicates that these mutant dimers are more flexible than the wild‐type protein and can form multiple cross‐linked dimeric species due to the transient exposure of cysteine residues that are inaccessible in the wild‐type protein. The enhanced flexibility of Glu18 DJ‐1 mutants provides a parsimonious explanation for their lower observed cross‐linking efficiency in cells. In addition, thiol cross‐linkers may have an underappreciated value as qualitative probes of protein conformational flexibility.