Methods: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels.Results: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes.Conclusions: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention.

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.

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-β 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00702-017-1726-7) contains supplementary material, which is available to authorized users.

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.

We recently hypothesized that parkin plays a role in redox homeostasis and provided evidence that it directly reduces hydrogen peroxide (H 2 O 2 ) in vitro. Here, we examined this anti-oxidant activity in vivo. Informed by findings in human brain, we demonstrate that elevated oxidative stress promotes parkin insolubility in mice. In normal mouse brain parkin was partially oxidized, e.g., at cysteines 195 and 252, which was augmented by oxidative stress. Although under basal conditions H 2 O 2 levels were unchanged in adult prkn -/brain, a parkin-dependent reduction of cytosolic H 2 O 2 was observed when mitochondria were impaired, either due to neurotoxicant exposure (MPTP) or Sod2 haploinsufficiency. In accordance, markers of oxidative stress, e.g., protein carbonylation and nitrotyrosination, were elevated in the cytosol but not in mitochondria from prkn -/mice. This rise in oxidative stress was associated with altered glutathione homeostasis. In parkin's absence reduced glutathione concentrations were increased in cells, murine brain and human cortex. This compensation was not due to new glutathione synthesis but attributed to elevated oxidized glutathione (GSSG)-reductase activity. Moreover, we discovered that parkin also recycled GSSG to its reduced form. With this reaction, parkin became S-glutathionylated, e.g., at cysteines 59 and human-specific 95. This oxidative modification was reversed by glutaredoxin. Our results demonstrate that cytosolic parkin mediates anti-oxidant reactions including H 2 O 2 reduction and glutathione regeneration.These reducing activities lead to a range of oxidative modifications in parkin itself. In parkin-deficient brain oxidative stress rises despite changes to maintain redox balance. wordsIn oxidative stress-exposed, human embryonic kidney (HEK293) cells, which transiently over-expressed PRKN, we also observed HMW smear formation. Parkin became progressively gel excluded (aggregated) and insoluble following exposure to higher levels of H 2 O 2 (Fig. 1b). Since these HMW forms of parkin were reversed with dithiothreitol (DTT; Extended Data Fig. 1a, c), we postulated that these modifications occurred due to increasing oxidation of parkin's cysteines 17 . We tested this using Nethylmaleimide (NEM) and iodoacetamide (IAA), which irreversibly alkylate free thiols.There, we found that pre-incubation of cells with NEM or IAA blocked HMW smear formation of parkin and preserved its solubility during H 2 O 2 stress (Fig. 1b; Extended Data Fig. 1b). Complementing these findings, we mapped several cysteines in recombinant (r-) parkin, which became oxidized when the protein was subjected to rising H 2 O 2 levels in vitro, thereby promoting its aggregation and insolubility 11 .Intriguingly, exposure to DTT also lowered the solubility of cellular parkin (Extended Data Fig. 1c), which suggested that parkin's structure is altered under excessive oxidizing and excessive reducing conditions. These results demonstrated that human parkin acts as a redox-sensitive molecule, which undergoes thio...

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, which are reflected in its posttranslational modifications. We found that in human control brain, including the S. nigra, parkin is largely insoluble after age 40 years, which is linked to its oxidation, e.g., at Cys95 and Cys253. In mice, oxidative stress increases posttranslational modifications at parkin cysteines and reduces its solubility. Oxidation of recombinant parkin also promotes insolubility and aggregate formation, but in parallel, lowers hydrogen peroxide (H2O2). This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. Intriguingly, in parkin-deficient human brain H2O2 concentrations are elevated. In prkn-null mice, H2O2 levels are dysregulated under oxidative stress conditions, such as acutely by MPTP-toxin exposure or chronically due to a second genetic hit. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic M17 cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at primate-specific Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation. In sections of normal, adult human midbrain, parkin specifically 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 redox effects may augment oxidative stress in dopamine producing neurons of mutant PRKN allele carriers, thereby contributing to neurodegeneration.

The mechanisms by which Parkinson disease-linked parkin confers neuroprotection of human dopamine cells remain elusive. We hypothesized that its cysteines mediate multiple anti-oxidant effects in the midbrain. By studying >60 control specimens, we found that in adult human brainbut not in skeletal muscle-parkin is mostly aggregated and insoluble due to oxidative modifications, such as at C253. In vitro, parkin's oxidation directly reduces hydrogen peroxide (H2O2) to water. In parkin-deficient human brain, H2O2 concentrations are elevated. In dopamine toxicity studies, wild-type parkin -but not disease-associated mutants-prevents neural death by lowering H2O2 and sequestering radicals within insoluble aggregates. Parkin conjugates dopamine metabolites at the human-specific residue C95 and augments melanin formation in vitro. Using epitope-mapped antibodies, we found that in adult Substantia nigra neurons parkin localizes to neuromelanin within LAMP-3/CD63-positive lysosomes. We conclude that parkin's own oxidation, previously considered a loss-of-function event, underlies three neuroprotective effects in adult midbrain: its cysteines participate in H2O2 reduction, dopamine radical conjugation and the formation of neuromelanin.Parkinson disease (PD) remains an incurable disorder of the human brain. Bi-allelic mutations in the PRKN gene, which encodes parkin, lead to young-onset, autosomal-recessive PD (ARPD) 1 . Clinicopathological studies of parkin-deficient brains have demonstrated restricted cell loss in the S. nigra and L. coeruleus, two nuclei synthesizing dopamine (DA). The field has not yet explained the selective neurodegeneration of PRKN-linked ARPD vs. other forms of PD.Parkin, a principally cytosolic protein, has been associated with diverse cellular functions, foremost related to ubiquitin ligase activity and mitochondrial integrity 2 . However, none of these roles has explained its neuroprotective selectivity, and animal models of genomic prkn deletion have failed to reproduce DA cell loss and parkinsonism. This observation could be due to compensatory mechanisms, a shorter life span, or the uniqueness of human DA metabolism. The latter is exemplified by the generation of neuromelanin in dopaminergic cells beginning in late childhood 3,4 . Nevertheless, select prkn-null models have revealed changes in high energy-producing cells of flies 5 and murine brain indicative of augmented oxidative stress [6][7][8] . We postulated that wild-type parkin promotes redox homeostasis in vivo.Redox equilibrium frequently involves cysteine-based chemistry; there, thiols are subject to oxidative modifications by reactive oxygen-, reactive nitrogen-and reactive electrophilic species (ROS, RNS, RES) 9 , some of which are reversible. Proteins irreversibly conjugated by RES, including by select DA radicals, are degraded, secreted or sequestered within inclusions. It is thought that this process underlies neuromelanin formation 10 .Human parkin contains 35 cysteines 1 . Several reports previously demonstrated its sensitivit...

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of alpha-Synuclein (a-Syn) protein. Currently, no treatment can slow nor halt neurodegeneration. Multiplications and mutations of the a-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress a-Syn replicate several features of PD. Decreasing total a-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with a 'synucleinopathy'. We previously performed a genetic screen for modifiers of a-Syn levels, and found CDK14, a kinase of largely unknown function as a regulator of a-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we decreased Cdk14 in two mouse models of synucleinopathy. We found that reduction of Cdk14 mitigated neuropathological and neurobehavioral sequelae associated with a-Syn overexpression. We further validated these findings in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable ex vivo. We found that in both mouse and human neurons, CDK14 inhibition decreases total and pathologically aggregated a-Syn. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.