DJ‐1 deficiency impairs autophagy and reduces alpha‐synuclein phagocytosis by microglia

Parkinson's disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ‐1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α‐Synuclein (α‐Syn) protein. Recent findings have shown that α‐Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α‐Syn. We previously reported that the knock down (KD) of DJ‐1 in microglia increased cells’ neurotoxicity to dopaminergic neurons. Here, we discovered that α‐Syn significantly induced elevated secretion of the proinflammatory cytokines IL‐6 and IL‐1β and a significant dose‐dependent elevation in the production of nitric oxide in DJ‐1 KD microglia, compared to control microglia. We further investigated the ability of DJ‐1 KD microglia to uptake and degrade soluble α‐Syn, and discovered that DJ‐1 KD reduces cell‐surface lipid raft expression in microglia and impairs their ability to uptake soluble α‐Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ‐1 KD microglia exhibit an impaired autophagy‐dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α‐Syn uptake and clearance in DJ‐1 KD microglia, compared to control microglia. Further studies of the link between DJ‐1, α‐Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.

     

Inhibition of mitochondrial fusion by α‐synuclein is rescued by PINK1, Parkin and DJ‐1

Aggregation of α‐synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age‐dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA‐mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ‐1 but not the PD‐associated mutations PINK1 G309D and parkin Δ1–79 or by DJ‐1 C106A.     

Transient sampling of aggregation‐prone conformations causes pathogenic instability of a parkinsonian mutant of DJ‐1 at physiological temperature

Various missense mutations in the cytoprotective protein DJ‐1 cause rare forms of inherited parkinsonism. One mutation, M26I, diminishes DJ‐1 protein levels in the cell but does not result in large changes in the three‐dimensional structure or thermal stability of the protein. Therefore, the molecular defect that results in loss of M26I DJ‐1 protective function is unclear. Using NMR spectroscopy near physiological temperature, we found that the picosecond–nanosecond dynamics of wild‐type and M26I DJ‐1 are similar. In contrast, elevated amide hydrogen/deuterium exchange rates indicate that M26I DJ‐1 is more flexible than the wild‐type protein on longer timescales and that hydrophobic regions of M26I DJ‐1 are transiently exposed to solvent. Tryptophan fluorescence spectroscopy and thiol crosslinking analyzed by mass spectrometry also demonstrate that M26I DJ‐1 samples conformations that differ from the wild‐type protein at 37°C. These transiently sampled conformations are unstable and cause M26I DJ‐1 to aggregate in vitro at physiological temperature but not at lower temperatures. M26I DJ‐1 aggregation is correlated with pathogenicity, as the structurally similar but non‐pathogenic M26L mutation does not aggregate at 37°C. The onset of dynamically driven M26I DJ‐1 instability at physiological temperature resolves conflicting literature reports about the behavior of this disease‐associated mutant and illustrates the pitfalls of characterizing proteins exclusively at room temperature or below, as key aspects of their behavior may not be apparent.     

RAC1 P29S regulates PD‐L1 expression in melanoma

Whole exome sequencing of cutaneous melanoma has led to the detection of P29 mutations in RAC1 in 5–9% of samples, but the role of RAC1 P29 mutations in melanoma biology remains unclear. Using reverse phase protein array analysis to examine the changes in protein/phospho‐protein expression, we identified cyclin B1, PD‐L1, Ets‐1, and Syk as being selectively upregulated with RAC1 P29S expression and downregulated with RAC1 P29S depletion. Using the melanoma patient samples in TCGA, we found PD‐L1 expression to be significantly increased in RAC1 P29S patients compared to RAC1 WT as well as other RAC1 mutants. The finding that PD‐L1 is upregulated suggests that oncogenic RAC1 P29S may promote suppression of the antitumor immune response. This is a new insight into the biological function of RAC1 P29S mutations with potential clinical implications as PD‐L1 is a candidate biomarker for increased benefit from treatment with anti‐PD1 or anti‐PD‐L1 antibodies.     

The minimal α‐crystallin domain of Mj Hsp16.5 is functional at non‐heat‐shock conditions

The small heat shock protein (sHSP) from Methanococcus jannaschii (Mj Hsp16.5) forms a monodisperse 24mer and each of its monomer contains two flexible N‐ and C‐terminals and a rigid α‐crystallin domain with an extruding β‐strand exchange loop. The minimal α‐crystallin domain with a β‐sandwich fold is conserved in sHSP family, while the presence of the β‐strand exchange loop is divergent. The function of the β‐strand exchange loop and the minimal α‐crystallin domain of Mj Hsp16.5 need further study. In the present study, we constructed two fragment‐deletion mutants of Mj Hsp16.5, one with both the N‐ and C‐terminals deleted (ΔNΔC) and the other with a further deletion of the β‐strand exchange loop (ΔNΔLΔC). ΔNΔC existed as a dimer in solution. In contrast, the minimal α‐crystallin domain ΔNΔLΔC became polydisperse in solution and exhibited more efficient chaperone‐like activities to prevent amorphous aggregation of insulin B chain and fibril formation of the amyloidogenic peptide dansyl‐SSTSAA‐W than the mutant ΔNΔC and the wild type did. The hydrophobic probe binding experiments indicated that ΔNΔLΔC exposed much more hydrophobic surface than ΔNΔC. Our study also demonstrated that Mj Hsp16.5 used different mechanisms for protecting different substrates. Though Mj Hsp16.5 formed stable complexes with substrates when preventing thermal aggregation, no complexes were detected when preventing aggregation under non‐heat‐shock conditions. Proteins 2014; 82:1156–1167. © 2013 Wiley Periodicals, Inc.     

DJ‐1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle‐like Cells in Response to Sphingosylphosphorylcholine

Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox‐sensitive molecules including a multifunctional protein DJ‐1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)‐stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α‐smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC‐stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ‐1 in hMSCs in response to SPC. The increased abundance of oxidized DJ‐1 in SPC‐stimulated hMSCs was validated by immunoblot analysis. The SPC‐induced increase in the expression of α‐SMA was stronger in DJ‐1‐knockdown hMSCs than in control cells. Moreover, the expression of α‐SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ‐1‐overexpressing hMSCs. Exogenous H₂O₂ mimicked the responses induced by SPC treatment. These results indicate that the ROS‐related DJ‐1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

DJ‐1 deficiency triggers microglia sensitivity to dopamine toward a pro‐inflammatory phenotype that is attenuated by rasagiline

DJ‐1 is an oxidative stress sensor that localizes to the mitochondria when the cell is exposed to oxidative stress. DJ‐1 mutations that result in gene deficiency are linked to increased risk of Parkinson's disease (PD). Activation of microglial stress conditions that are linked to PD may result in neuronal death. We postulated that DJ‐1 deficiency may increase microglial neurotoxicity. We found that down‐regulation of DJ‐1 in microglia using an shRNA approach increased cell sensitivity to dopamine as measured by secreted pro‐inflammatory cytokines such as IL‐1β and IL‐6. Furthermore, we discovered that DJ‐1‐deficient microglia had increased monoamine oxidase activity that resulted in elevation of intracellular reactive oxygen species and nitric oxide leading to increased dopaminergic neurotoxicity. Rasagaline, a monoamine oxidase inhibitor approved for treatment of PD, reduced the microglial pro‐inflammatory phenotype and significantly reduced neurotoxicity. Moreover, we discovered that DJ‐1‐deficient microglia have reduced expression of triggering receptor expressed on myeloid cells 2 (TREM2), previously suggested as a risk factor for pro‐inflammation in neurodegenerative diseases. Further studies of DJ‐1‐mediated cellular pathways in microglia may contribute useful insights into the development of PD providing future avenues for therapeutic intervention.

     

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 protein's 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.

     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

α‐MSH activates immediate defense responses to UV‐induced oxidative stress in human melanocytes

Exposure of cultured human melanocytes to ultraviolet radiation (UV) results in DNA damage. In melanoma, UV‐signature mutations resulting from unrepaired photoproducts are rare, suggesting the possible involvement of oxidative DNA damage in melanocyte malignant transformation. Here we present data demonstrating immediate dose‐dependent generation of hydrogen peroxide in UV‐irradiated melanocytes, which correlated directly with a decrease in catalase activity. Pretreatment of melanocytes with α‐melanocortin (α‐MSH) reduced the UV‐induced generation of 7,8‐dihydro‐8‐oxyguanine (8‐oxodG), a major form of oxidative DNA damage. Pretreatment with α‐MSH also increased the protein levels of catalase and ferritin. The effect of α‐MSH on 8‐oxodG induction was mediated by activation of the melanocortin 1 receptor (MC1R), as it was absent in melanocytes expressing loss‐of‐function MC1R, and blocked by concomitant treatment with an analog of agouti signaling protein (ASIP), ASIP‐YY. This study provides unequivocal evidence for induction of oxidative DNA damage by UV in human melanocytes and reduction of this damage by α‐MSH. Our data unravel some mechanisms by which α‐MSH protects melanocytes from oxidative DNA damage, which partially explain the strong association of loss‐of‐function MC1R with melanoma.     

Analysis of two benzo[a]pyrene-resistant mutants of the mouse hepatoma Hepa-1 P(1)450 gene via cDNA expression in yeast.

Two benzo[a]pyrene-resistant mutant clones (c1 and c37) of the mouse hepatoma Hepa-1 wild-type (wt) cell line were examined for their lack of P(1)450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)] activity. From lambda gt11 cDNA libraries, the nearly full-length P(1)450 cDNAs of wt, c1 and c37 were isolated and sequenced. The c1 cDNA was found to have a single mutation leading to premature termination of the protein after Asn-414; a rapidly migrating band corresponding to this truncated protein was found on Western immunoblots. The c37 cDNA was found to have two point mutations, leading to Leu-118----Arg-118 and Arg-245----Pro-245, but otherwise to encode the normal (524-residue) protein; the mature protein was confirmed by Western blot analysis. P(1)450 cDNA from wt, c1 and c37 and chimeric cDNAs between wt and c37 were inserted into the expression vector pAAH5 and expressed in Saccharomyces cerevisiae strain 50.L4. The Leu-118----Arg-118 mutation alone was found to have negligible effect on AHH activity, while the Arg-245----Pro-245 mutation alone leads to a 2- to 3-fold decrease in enzyme activity. The two mutations together totally abrogate AHH activity. The biologic mutant c37 provides the first evidence for the importance of Arg-245, and the complementary function of Leu-118, in normal P(1)450 enzymic function. This alteration in a single amino acid from arginine to proline might block electron flow directly, or change secondary structure of the protein, such that normal monooxygenation of benzo[a]pyrene cannot occur.     

Stable overexpression of DJ‐1 protects H9c2 cells against oxidative stress under a hypoxia condition

It has been well accepted that increased reactive oxygen species (ROS) and the subsequent oxidative stress is one of the major causes of ischemia/reperfusion (I/R) injury. DJ‐1 protein, as a multifunctional intracellular protein, plays an important role in regulating cell survival and antioxidant stress. Here, we wondered whether DJ‐1 overexpression attenuates simulated ischemia/reperfusion (sI/R)‐induced oxidative stress. A rat cDNA encoding DJ‐1 was inserted into a mammalian expression vector. After introduction of this construct into H9c2 myocytes, stable clones were obtained. Western blot analysis of the derived clones showed a 2.6‐fold increase in DJ‐1 protein expressing. Subsequently, the DJ‐1 gene‐transfected and control H9c2 cells were subjected to sI/R, and then cell viability, lactate dehydrogenase, malondialdehyde, intracellular ROS and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were measured appropriately. The results showed that stable overexpression of DJ‐1 efficiently attenuated sI/R‐induced viability loss and lactate dehydrogenase leakage. Additionally, stable overexpression of DJ‐1 inhibited sI/R‐induced the elevation of ROS and MDA contents followed by the increase of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities and expression. Our data indicate that overexpression of DJ‐1 attenuates ROS generation, enhances the cellular antioxidant capacity and prevents sI/R‐induced oxidative stress, revealing a novel mechanism of cardioprotection. Importantly, DJ‐1 overexpression may be an important part of a protective strategy against ischemia/reperfusion injury. Copyright © 2012 John Wiley & Sons, Ltd.     

α‐secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Aβ generation

The Swedish mutation within the amyloid precursor protein (APP) causes early‐onset Alzheimer’s disease due to increased cleavage of APP by BACE1. While β‐secretase shedding of Swedish APP (APPswe) largely results from an activity localized in the late secretory pathway, cleavage of wild‐type APP occurs mainly in endocytic compartments. However, we show that liberation of Aβ from APPswe is still dependent on functional internalization from the cell surface. Inspite the unchanged overall β‐secretase cleaved soluble APP released from APP_swe secretion, mutations of the APPswe internalization motif strongly reduced C99 levels and substantially decreased Aβ secretion. We point out that α‐secretase activity‐mediated conversion of C99 to C83 is the main cause of this Aβ reduction. Furthermore, we demonstrate that α‐secretase cleavage of C99 even contributes to the reduction of Aβ secretion of internalization deficient wild‐type APP. Therefore, inhibition of α‐secretase cleavage increased Aβ secretion through diminished conversion of C99 to C83 in APP695, APP695swe or C99 expressing cells.     

Choline transporter‐like1 (CHER1) is crucial for plasmodesmata maturation in Arabidopsis thaliana

Plasmodesmata (PD) are microscopic pores connecting plant cells and enable cell‐to‐cell transport. Currently, little information is known about the molecular mechanisms regulating PD formation and development. To uncover components of PD development we made use of the 17 kDa movement protein (MP17) encoded by the Potato leafroll virus (PLRV). The protein is required for cell‐to‐cell movement of the virus and localises to complex PD. Forward genetic screening for Arabidopsis mutants with altered PD binding of MP17 revealed several mutant lines, while molecular genetics, biochemical and microscopic studies allowed further characterisation. Map‐based cloning of one mutant revealed a point mutation in the choline transporter‐like 1 (CHER1) protein, changing glycine²⁴⁷ into glutamate. Mutation in CHER1 resulted in a starch excess phenotype and stunted growth. Ultrastructure analysis of shoot apical meristems, developing and fully developed leaves showed reduced PD numbers and the absence of complex PD in fully developed leaves. This indicates that cher1 mutants are impaired in PD formation and development. Global lipid profiling revealed only slight modifications in the overall lipid composition, however, altered composition of PD‐associated lipids cannot be ruled out. Thus, cher1 is devoid of complex PD in developed leaves and provides insights into the formation of complex PD at the molecular level.     

Differential regulation of NMDA receptor function by DJ‐1 and PINK1

Dysfunction of PTEN‐induced kinase 1 (PINK1) or DJ‐1 promotes neuronal death and is implicated in the pathogenesis of Parkinson’s disease, but the underlying mechanisms remain unclear. Given the roles of N‐methyl‐d‐ aspartate receptor (NMDAr)‐mediated neurotoxicity in various brain disorders including cerebral ischemia and neurodegenerative diseases, we investigated the effects of PINK1 and DJ‐1 on NMDAr function. Using protein overexpression and knockdown approaches, we showed that PINK1 increased NMDAr‐mediated whole‐cell currents by enhancing the function of NR2A‐containing NMDAr subtype (NR2ACNR). However, DJ‐1 decreased NMDAr‐mediated currents, which was mediated through the inhibition of both NR2ACNR and NR2B‐containing NMDAr subtype (NR2BCNR). We revealed that the knockdown of DJ‐1 enhanced PTEN expression, which not only potentiated NR2BCNR function but also increased PINK1 expression that led to NR2ACNR potentiation. These results indicate that NMDAr function is differentially regulated by DJ‐1‐dependent signal pathways DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR. Our results further showed that the suppression of DJ‐1, while promoted NMDA‐induced neuronal death through the overactivation of PTEN/NR2BCNR‐dependent cell death pathway, induced a neuroprotective effect to counteract DJ‐1 dysfunction‐mediated neuronal death signaling through activating PTEN/PINK1/NR2ACNR cell survival–promoting pathway. Thus, PINK1 acts with DJ‐1 in a common pathway to regulate NMDAr‐mediated neuronal death. This study suggests that the DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR pathways may represent potential therapeutic targets for the development of neuroprotection strategy in the treatment of brain injuries and neurodegenerative diseases such as Parkinson’s disease.