Age-associated insolubility of parkin in human midbrain is linked to redox balance and sequestration of reactive dopamine metabolites.

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson's-linked neurodegeneration.

Modelling idiopathic Parkinson disease as a complex illness can inform incidence rate in healthy adults: the PR EDIGT score.

Fifty-five years after the concept of dopamine replacement therapy was introduced, Parkinson disease (PD) remains an incurable neurological disorder. To date, no disease-modifying therapeutic has been approved. The inability to predict PD incidence risk in healthy adults is seen as a limitation in drug development, because by the time of clinical diagnosis ≥ 60% of dopamine neurons have been lost. We have designed an incidence prediction model founded on the concept that the pathogenesis of PD is similar to that of many disorders observed in ageing humans, i.e. a complex, multifactorial disease. Our model considers five factors to determine cumulative incidence rates for PD in healthy adults: (i) DNA variants that alter susceptibility (D), e.g. carrying a LRRK2 or GBA risk allele; (ii) Exposure history to select environmental factors including xenobiotics (E); (iii) Gene-environment interactions that initiate pathological tissue responses (I), e.g. a rise in ROS levels, misprocessing of amyloidogenic proteins (foremost, α-synuclein) and dysregulated inflammation; (iv) sex (or gender; G); and importantly, (v) time (T) encompassing ageing-related changes, latency of illness and propagation of disease. We propose that cumulative incidence rates for PD (PR ) can be calculated in healthy adults, using the formula: PR (%) = (E + D + I) × G × T. Here, we demonstrate six case scenarios leading to young-onset parkinsonism (n = 3) and late-onset PD (n = 3). Further development and validation of this prediction model and its scoring system promise to improve subject recruitment in future intervention trials. Such efforts will be aimed at disease prevention through targeted selection of healthy individuals with a higher prediction score for developing PD in the future and at disease modification in subjects that already manifest prodromal signs.

Holocranohistochemistry enables the visualization of α-synuclein expression in the murine olfactory system and discovery of its systemic anti-microbial effects.

Braak and Del Tredici have proposed that typical Parkinson disease (PD) has its origins in the olfactory bulb and gastrointestinal tract. However, the role of the olfactory system has insufficiently been explored in the pathogeneses of PD and Alzheimer disease (AD) in laboratory models. Here, we demonstrate applications of a new method to process mouse heads for microscopy by sectioning, mounting, and staining whole skulls ('holocranohistochemistry'). This technique permits the visualization of the olfactory system from the nasal cavity to mitral cells and dopamine-producing interneurons of glomeruli in the olfactory bulb. We applied this method to two specific goals: first, to visualize PD- and AD-linked gene expression in the olfactory system, where we detected abundant, endogenous α-synuclein and tau expression in the olfactory epithelium. Furthermore, we observed amyloid-ß plaques and proteinase-K-resistant α-synuclein species, respectively, in cranial nerve-I of APP- and human SNCA-over-expressing mice. The second application of the technique was to the modeling of gene-environment interactions in the nasal cavity of mice. We tracked the infection of a neurotropic respiratory-enteric-orphan virus from the nose pad into cranial nerves-I (and -V) and monitored the ensuing brain infection. Given its abundance in the olfactory epithelia, we questioned whether α-synuclein played a role in innate host defenses to modify the outcome of infections. Indeed, Snca-null mice were more likely to succumb to viral encephalitis versus their wild-type littermates. Moreover, using a bacterial sepsis model, Snca-null mice were less able to control infection after intravenous inoculation with Salmonella typhimurium. Together, holocranohistochemistry enabled new discoveries related to α-synuclein expression and its function in mice. Future studies will address: the role of Mapt and mutant SNCA alleles in infection paradigms; the contribution of xenobiotics in the initiation of idiopathic PD; and the safety to the host when systemically targeting α-synuclein by immunotherapy.

CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing.

Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.

Type-I interferon signaling through ISGF3 complex is required for sustained Rip3 activation and necroptosis in macrophages.

Myeloid cells play a critical role in perpetuating inflammation during various chronic diseases. Recently the death of macrophages through programmed necrosis (necroptosis) has emerged as an important mechanism in inflammation and pathology. We evaluated the mechanisms that lead to the induction of necrotic cell death in macrophages. Our results indicate that type I IFN (IFN-I) signaling is a predominant mechanism of necroptosis, because macrophages deficient in IFN-α receptor type I (IFNAR1) are highly resistant to necroptosis after stimulation with LPS, polyinosinic-polycytidylic acid, TNF-α, or IFN-ß in the presence of caspase inhibitors. IFN-I-induced necroptosis occurred through both mechanisms dependent on and independent of Toll/IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) and led to persistent phosphorylation of receptor-interacting protein 3 (Rip3) kinase, which resulted in potent necroptosis. Although various IFN-regulatory factors (IRFs) facilitated the induction of necroptosis in response to IFN-ß, IRF-9-STAT1- or -STAT2-deficient macrophages were highly resistant to necroptosis. Our results indicate that IFN-ß-induced necroptosis of macrophages proceeds through tonic IFN-stimulated gene factor 3 (ISGF3) signaling, which leads to persistent expression of STAT1, STAT2, and IRF9. Induction of IFNAR1/Rip3-dependent necroptosis also resulted in potent inflammatory pathology in vivo. These results reveal how IFN-I mediates acute inflammation through macrophage necroptosis.

Cathepsins limit macrophage necroptosis through cleavage of Rip1 kinase.

It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells.

Parkin coregulates glutathione metabolism in adult mammalian brain.

We recently discovered that the expression of PRKN, a young-onset Parkinson disease-linked gene, confers redox homeostasis. To further examine the protective effects of parkin in an oxidative stress model, we first combined the loss of prkn with Sod2 haploinsufficiency in mice. Although adult prkn-/-//Sod2± animals did not develop dopamine cell loss in the S. nigra, they had more reactive oxidative species and a higher concentration of carbonylated proteins in the brain; bi-genic mice also showed a trend for more nitrotyrosinated proteins. Because these redox changes were seen in the cytosol rather than mitochondria, we next explored the thiol network in the context of PRKN expression. We detected a parkin deficiency-associated increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in murine brain, PRKN-linked human cortex and several cell models. This shift resulted from enhanced recycling of GSSG back to GSH via upregulated glutathione reductase activity; it also correlated with altered activities of redox-sensitive enzymes in mitochondria isolated from mouse brain (e.g., aconitase-2; creatine kinase). Intriguingly, human parkin itself showed glutathione-recycling activity in vitro and in cells: For each GSSG dipeptide encountered, parkin regenerated one GSH molecule and was S-glutathionylated by the other (GSSG + P-SH [Formula: see text] GSH + P-S-SG), including at cysteines 59, 95 and 377. Moreover, parkin's S-glutathionylation was reversible by glutaredoxin activity. In summary, we found that PRKN gene expression contributes to the network of available thiols in the cell, including by parkin's participation in glutathione recycling, which involves a reversible, posttranslational modification at select cysteines. Further, parkin's impact on redox homeostasis in the cytosol can affect enzyme activities elsewhere, such as in mitochondria. We posit that antioxidant functions of parkin may explain many of its previously described, protective effects in vertebrates and invertebrates that are unrelated to E3 ligase activity.

Transcriptional regulation of the Yts1 type II secretion system of Yersinia enterocolitica and identification of secretion substrates.

Type II secretion systems (T2SSs) mediate the transport of proteins through the bacterial outer membrane. Although widespread in gamma-proteobacteria, they have so far not been characterized in the human pathogen Yersinia enterocolitica. We describe here genetic linkage of the Yts1 and Yts2 T2SSs with a transcriptional regulator in the highly pathogenic Y. enterocolitica biotype 1B. While the Yts2-associated PypC regulator activates the Yts2 operon, the PclR regulator does not induce transcription of its cognate Yts1 operon. Instead, Yts1 and pclR are activated by the addition of MgCl(2) to the growth medium at 17 degrees C and 26 degrees C, but not at 37 degrees C. We identified three proteins, ChiY, EngY (YE2830) and YE3650, that are secreted depending on a functional Yts1 T2SS in response to MgCl(2) at low temperature. While the activation of chiY by MgCl(2) depends on pclR, PclR overproduction is not sufficient for chiY transcription in an Escherichia coli background, demonstrating the need for additional Y. enterocolitica-specific factors. As ChiY and EngY both bind to chitin, and YE3650 shows motifs conserved in oligosaccharide-binding enzymes, all secreted proteins might be important for polysaccharide interaction/degradation during infection or in the environment.

Ripk3 licenced protection against microbial infection in the absence of Caspase1-11 inflammasome.

Receptor interacting protein kinase 3 (Ripk3) is a signal relay protein involved in initiation of programmed cell death (necroptosis) and modulation of inflammasome activation. While caspase 1 and 11 are pro-inflammatory caspases responsible for unleashing inflammation and cell death by enzymatic activation of the executioners of inflammation and cell death (pyroptosis). Upon Salmonella infection, the host mounts a pro-inflammatory response which require Ripk3 and Caspase1/11. Here we show that bone marrow derived macrophages with combined deficiency of Ripk3 and Casp1/11 are highly resistant to Salmonella induced cell death, and that these macrophages show an anti-inflammatory cytokine profile. We confirm what was previously known that mice deficient in Casp1/11 have impaired ability to control Salmonella burden, and that the absence of Ripk3 alone does not influence the innate immune responses to Salmonella infection. However, we describe a synergistic role of Ripk3 and Casp1/11 in regulating Salmonella in vivo burden and that Ripk3-dependent host protection in the absence of Casp1/11 is evident during infection by sifA-expressing Salmonella. In summary, we show that the Ripk3 protection to Salmonella infection is obscured by presence of Caspase 1/11 and that the Ripk3-dependent protection requires a phagosome-bound Salmonella.

Lrrk2 alleles modulate inflammation during microbial infection of mice in a sex-dependent manner.

Variants in the leucine-rich repeat kinase-2 (LRRK2) gene are associated with Parkinson's disease, leprosy, and Crohn's disease, three disorders with inflammation as an important component. Because of its high expression in granulocytes and CD68-positive cells, LRRK2 may have a function in innate immunity. We tested this hypothesis in two ways. First, adult mice were intravenously inoculated with Salmonella typhimurium, resulting in sepsis. Second, newborn mouse pups were intranasally infected with reovirus (serotype 3 Dearing), which induced encephalitis. In both mouse models, wild-type Lrrk2 expression was protective and showed a sex effect, with female Lrrk2-deficient animals not controlling infection as well as males. Mice expressing Lrrk2 carrying the Parkinson's disease-linked p.G2019S mutation controlled infection better, with reduced bacterial growth and longer animal survival during sepsis. This gain-of-function effect conferred by the p.G2019S mutation was mediated by myeloid cells and was abolished in animals expressing a kinase-dead Lrrk2 variant, p.D1994S. Mouse pups with reovirus-induced encephalitis that expressed the p.G2019S Lrrk2 mutation showed increased mortality despite lower viral titers. The p.G2019S mutant Lrrk2 augmented immune cell chemotaxis and generated more reactive oxygen species during virulent infection. Reovirus-infected brains from mice expressing the p.G2019S mutant Lrrk2 contained higher concentrations of α-synuclein. Animals expressing one or two p.D1994S Lrrk2 alleles showed lower mortality from reovirus-induced encephalitis. Thus, Lrrk2 alleles may alter the course of microbial infections by modulating inflammation, and this may be dependent on the sex and genotype of the host as well as the type of pathogen.

Culling of APCs by inflammatory cell death pathways restricts TIM3 and PD-1 expression and promotes the survival of primed CD8 T cells.

We evaluated the impact of premature cell death of antigen-presenting cells (APCs) by Caspase-1- and RipK3-signaling pathways on CD8+ T-cell priming during infection of mice with Salmonella typhimurium (ST). Our results indicate that Caspase1 and RipK3 synergize to rapidly eliminate infected APCs, which does not influence the initial activation of CD8+ T cells. However, the maintenance of primed CD8+ T cells was greatly compromised when both these pathways were disabled. Caspase-1- and RipK3-signaling did not influence NF-κB signaling in APCs, but synergized to promote processing of IL-1 and IL-18. Combined deficiency of Caspase1 and RipK3 resulted in compromised innate immunity and accelerated host fatality due to poor processing of IL-18. In contrast, synergism in cell death by Caspase-1- and RipK3 resulted in restriction of PD-1 and TIM3 expression on primed CD8+ T cells, which promoted the survival of activated CD8+ T cells.

Controlled activation of the Cpx system is essential for growth of Yersinia enterocolitica.

The Cpx two-component signal transduction system regulates the expression of genes in response to stimuli associated with the maintenance of the bacterial cell envelope. Additionally, the Cpx system is important for virulence in several bacterial pathogens. In this study, we analyzed the Cpx system of the human enteropathogen Yersinia enterocolitica. We provide evidence that transcription of cpxR is autoregulated and that the response regulator CpxR negatively affects transcription of rpoE, coding for the extracytoplasmic function sigma factor sigma(E), thereby linking at the transcriptional level two systems involved in envelope maintenance. A mutated form of CpxR that cannot be phosphorylated, CpxRD51A, affects transcription of cpxR and rpoE similar to the wild-type CpxR. In contrast, CpxR and CpxRD51A differentially regulate transcription of htrA, indicating phosphorylation-dependent and -independent mechanisms of transcriptional regulation. Moreover, overproduction of CpxR, CpxRD51A and CpxA result in a growth defect. Interestingly, a cpxA mutant strain could not be obtained. We conclude that the phosphorylation status of CpxR needs to be tightly controlled by CpxA, and that the Cpx system has a central role in regulating basic cellular functions. In addition, we show that cpxR and cpxP mutant strains have no defect in Y. enterocolitica invasion of host cells.