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.

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.

Constructing and evaluating the effect of micron-scale structures on von Willebrand factor damage.

The incidence of clinical complication gastrointestinal bleeding has been proved as consequence of von Willebrand factor (VWF) damage after mechanical circulatory support in clinic. Many studies have been conducted to evaluate VWF damage, of which the most studied influencing factors are mechanical factors such as shear stress. However, in addition to mechanical factors, VWF damage may also be affected by interface factors. To address this issue, a roller pump circulation platform was established to investigate the effect of material surface micron-scale structures distribution on VWF damage in flow state. A composite micro-structure combining microngrating and micronpost was designed and constructed on the surface of Si wafer by lithography and reactive ion etching, and detailed characterization of material surfaces was also performed. Then the changes of VWF antigen, VWF ristocetin cofactor activity, and the degradation of high molecular weight VWF on these surfaces were investigated and compared. The results showed that, with the encryption of surface micro-structures arrangement, the material surface tends to be more hydrophobic, which is beneficial to reduce VWF damage. Therefore, in the design of material surface inside the mechanical circulatory support devices, it can be considered to add some surface micro-structures with a certain distribution density to change the hydrophilicity and hydrophobicity, so as to minimize the VWF damage. These results can provide important references for the evaluation of VWF damage caused by interface factors, and aid in designing material surface inside the mechanical circulatory support devices.

In vitro comparative study of red blood cell and VWF damage on 3D printing biomaterials under different blood-contacting conditions.

Mechanical circulatory support devices (MCSDs) are often associated with hemocompatible complications such as hemolysis and gastrointestinal bleeding when treating patients with end-stage heart failure. Shear stress and exposure time have been identified as the two most important mechanical factors causing blood damage. However, the materials of MCSDs may also induce blood damage when contacting with blood. In this study, the red blood cell and von Willebrand Factor (VWF) damage caused by four 3D printing biomaterials were investigated, including acrylic, PCISO, Somos EvoLVe 128, and stainless steel. A roller pump circulation experimental platform and a rotor blood-shearing experimental platform were constructed to mimic static and dynamic blood-contacting conditions of materials in MCSDs, respectively. Free hemoglobin assay and VWF molecular weight analysis were performed on the experimental blood samples. It indicated that different 3D printing materials and technology could induce different levels of damage to red blood cells and VWF, with acrylic causing the least damage under both static and dynamic conditions. In addition, it was found that blood damage measured for the same material differed on the two platforms. Therefore, a combination of static and dynamic experiments should be used to comprehensively investigate the effects of blood damage caused by the material. It can provide a reference for the design and evaluation of materials in different components of MCSDs.

A comprehensive comparison of the in vitro hemocompatibility of extracorporeal centrifugal blood pumps.

Purpose: Blood damage has been associated with patients under temporary continuous-flow mechanical circulatory support. To evaluate the side effects caused by transit blood pumping, in vitro hemocompatibility testing for blood damage in pumps is considered a necessary reference before clinical trials. Methods: The hemocompatibility of five extracorporeal centrifugal blood pumps was investigated comprehensively, including four commercial pumps (the Abbott CentriMag, the Terumo Capiox, the Medos DP3, and the Medtronic BPX-80) and a pump in development (the magAssist MoyoAssist®). In vitro, hemolysis was tested with heparinized porcine blood at nominal operating conditions (5 L/min, 160 mmHg) and extreme operating conditions (1 L/min, 290 mmHg) using a circulation flow loop. Hematology analyses concerning the blood cell counts and the degradation of high-molecular-weight von Willebrand factor (VWF) during 6-h circulation were also evaluated. Results: Comparing the in vitro hemocompatibility of blood pumps at different operations, the blood damage was significantly more severe at extreme operating conditions than that at nominal operating conditions. The performance of the five blood pumps was arranged in different orders at these two operating conditions. The results also demonstrated superior hemocompatibility of CentriMag and MoyoAssist® at two operating conditions, with overall low blood damage at hemolysis level, blood cell counts, and degradation of high-molecular-weight VWF. It suggested that magnetic bearings have an advantage in hemocompatibility compared to the mechanical bearing of blood pumps. Conclusion: Involving multiple operating conditions of blood pumps in in vitro hemocompatibility evaluation will be helpful for clinical application. In addition, the magnetically levitated centrifugal blood pump MoyoAssist® shows great potential in the future as it demonstrated good in vitro hemocompatibility.

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.

Spatiotemporal distribution of spectrin breakdown products induced by anoxia in adult rat optic nerve in vitro.

Hypoxic/ischemic and traumatic injury to central nervous system myelinated axons is heavily dependent on accumulation of Ca ions in the axoplasm, itself promoted by Na influx from the extracellular space. Given the high density of nodal Na channels, we hypothesized that nodes of Ranvier might be particularly vulnerable to Ca overload and subsequent damage, as this is the expected locus of maximal Na influx. Adult rat optic nerves were exposed to in vitro anoxia and analyzed immunohistochemically for the presence of spectrin breakdown. Cleavage of spectrin became detectable between 15 and 30 mins of anoxia, and increased homogeneously along the lengths of fibers; localized breakdown was not observed at nodes of Ranvier at any time point analyzed. Spectrin breakdown was also found in glial processes surrounding axons. Confocal imaging of axoplasmic Ca also revealed a gradual and nonlocalized increase as anoxia progressed, without evidence of Ca 'hot-spots' anywhere along the axons at any time between 0 and 30 mins of anoxic exposure in vitro. Calculations of Ca diffusion rates indicated that even if Ca entered or was released focally in axons, this ion would diffuse rapidly into the internodes and likely produce diffuse injury by activating Ca-dependent proteases. Western blot analysis for voltage-gated Na channel protein revealed that key functional proteins such as these are also degraded by anoxia/ischemia. Thus, proteolysis of structural and functional proteins will conspire to irreversibly injure central axons and render them nonfunctional, eventually leading to transection, degradation, and Wallerian degeneration.

Calpain-dependent neurofilament breakdown in anoxic and ischemic rat central axons.

Neurofilaments are key structural components of white matter axons. The effect of in vitro anoxia or oxygen-glucose deprivation (OGD) on the integrity of the 160 and 200 kDa neurofilament isoforms was studied by immunoblot, and correlated with physiological function. Adult rat optic nerves were exposed to 60 min of either anoxia or OGD. Compound action potential area recovered to 22+/-6% of control after 60 min of anoxia, and to 4+/-1% after 60 min of OGD. Ca(2+)-free (+EGTA) perfusate allowed complete recovery after OGD (108+/-42%). Tetrodotoxin (TTX, 1 microM) was less protective (45+/-6%). Both anoxia and OGD induced breakdown of neurofilament 160 (NF160) and NF200 revealed by the appearance of multiple lower molecular weight bands mainly in the 75-100 kDa range. Zero-Ca(2+)/EGTA completely prevented NF breakdown. TTX only partially reduced NF160 degradation. Non-phosphorylated NF200 appeared after reperfusion post-anoxia or OGD, and was also greatly reduced by zero-Ca(2+) or TTX. Calpain inhibitors (10 microM calpain inhibitor I or 50 microM MDL 28,170) significantly reduced NF160 and NF200 breakdown/dephosphorylation, but did not improve electrophysiological recovery. Significant calpain-mediated breakdown of NF160 and NF200 indicates structural damage to the axonal cytoskeleton, which was completely Ca(2+)-dependent. While pharmacological inhibition of calpain alone greatly reduced NF proteolysis, there was no concomitant improvement in function. These results imply that calpain inhibition is necessary but not sufficient for white matter protection, and emphasize the existence of multiple Ca(2+)-dependent degradative pathways activated in injured white matter.