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Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN-/- deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN-/- deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN-/- mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.

Upregulations of neuronal nitric oxide synthase (nNOS) in the rodent brain have been associated with neuronal aging. To address underlying mechanisms we generated SH-SY5Y neuronal cells constitutively expressing nNOS at a level similar to mouse brain (nNOS+ versus MOCK). Initial experiments revealed S-nitrosylations (SNO) of key players of protein homeostasis: heat shock cognate HSC70/HSPA8 within its nucleotide-binding site, and UBE2D ubiquitin conjugating enzymes at the catalytic site cysteine. HSPA8 is involved in protein folding, organelle import/export and chaperone-mediated LAMP2a-dependent autophagy (CMA). A set of deep redox and full proteome analyses, plus analysis of autophagy, CMA and ubiquitination with rapamycin and starvation as stimuli confirmed the initial observations and revealed a substantial increase of SNO modifications in nNOS+ cells, in particular targeting protein networks involved in protein catabolism, ubiquitination, carbohydrate metabolism and cell cycle control. Importantly, NO-independent reversible oxidations similarly occurred in both cell lines. Functionally, nNOS caused an accumulation of proteins, including CMA substrates and loss of LAMP2a. UBE2D activity and proteasome activity were impaired, resulting in dysregulations of cell cycle checkpoint proteins. The observed changes of protein degradation pathways caused an expansion of the cytoplasm, large lysosomes, slowing of the cell cycle and suppression of proliferation suggesting a switch of the phenotype towards aging, supported by downregulations of neuronal progenitor markers but increase of senescence-associated proteins. Hence, upregulation of nNOS in neuronal cells imposes aging by SNOing of key players of ubiquitination, chaperones and of substrate proteins leading to interference with crucial steps of protein homeostasis. Keywords: Redox modification, Nitric oxide, Autophagy, Ubiquitin, Chaperone, Lysosome, Posttranslational modification, Starvation, Rapamycin, Senescence

Upregulations of neuronal nitric oxide synthase (nNOS/NOS1) in the mouse brain upon aging suggest a role in age-associated changes of protein homeostasis. We generated a cell model, in which constitutive expression of nNOS in SH-SY5Y cells at a level comparable to mouse brain replicates the aging phenotype i.e. slowing of cell proliferation, cell enlargement and expression of senescence markers. nNOS+ and MOCK cells were exposed to proteostasis stress by treatment with rapamycin or serum-free starvation. The proteomes were analyzed per SILAC or label-free using hybrid liquid chromatography/mass spectrometry (LC/MS). Full scan MS-data were acquired using Xcalibur, and raw mass spectra were analyzed using the proteomics software MaxQuant. The human reference proteome from uniprot was used as template to identify peptides and proteins and quantify protein expression. The DiB data file contains essential MaxQuant output tables and includes peptide and protein identification, accession numbers, protein and gene names, sequence coverage and quantification values of each sample. Differences in protein expression in MOCK versus nNOS+ SH-SY5Y cells and interpretation of results are presented in Valek et al. (2018). Raw mass spectra and MaxQuant output files have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014) via the PRIDE partner repository with the dataset identifier PRIDE: PXD010538.

Upregulations of neuronal nitric oxide synthase (nNOS/NOS1) in the mouse brain upon aging and stress suggest a role of NO-dependent redox protein modifications for age-associated protein imbalances or dysfunctions. We generated a cell model, in which constitutive expression of nNOS in SH-SY5Y cells at a level comparable with mouse brain replicates the aging phenotype, that is, slowing of cell proliferation, cell enlargement, and expression of senescence markers. nNOS+ and MOCK cells were exposed to proteostasis stress by the treatment with rapamycin or serum-free starvation versus control conditions. To analyze NO-mediated S-nitrosylations (SNO) and other reversible protein modifications including disulfides and sulfoxides, we used complimentary proteomic approaches encompassing 2D-SNO-DIGE (differential gel electrophoresis), SNO-site identification (SNOSID), SNO Super-SILAC, SNO BIAM-Switch, and Redox-BIAM switch. The redox proteomes were analyzed using hybrid liquid chromatography/mass spectrometry (LC/MS). Full scan MS-data were acquired using Xcalibur, and raw mass spectra were analyzed using the proteomics software MaxQuant. The human reference proteome sets from uniprot were used as templates to identify peptides and proteins and quantify protein expression. The DiB data file contains MaxQuant output tables of the redox-modified proteins.The tables include peptide and protein identification, accession numbers, protein, and gene names, sequence coverage and quantification values of each sample. Differences in protein redox modifications in MOCK versus nNOS+ SH-SY5Y cells and interpretation of results are presented in (Valek et al., 2018).

Abstract Background PINK1 deficiency causes the autosomal recessive PARK6 variant of Parkinson’s disease. PINK1 activates ubiquitin by phosphorylation and cooperates with the downstream ubiquitin ligase PARKIN, to exert quality control and control autophagic degradation of mitochondria and of misfolded proteins in all cell types. Methods Global transcriptome profiling of mouse brain and neuron cultures were assessed in protein-protein interaction diagrams and by pathway enrichment algorithms. Validation by quantitative reverse transcriptase polymerase chain reaction and immunoblots was performed, including human neuroblastoma cells and patient primary skin fibroblasts. Results In a first approach, we documented Pink1-deleted mice across the lifespan regarding brain mRNAs. The expression changes were always subtle, consistently affecting “intracellular membrane-bounded organelles”. Significant anomalies involved about 250 factors at age 6 weeks, 1300 at 6 months, and more than 3500 at age 18 months in the cerebellar tissue, including Srsf10, Ube3a, Mapk8, Creb3, and Nfkbia. Initially, mildly significant pathway enrichment for the spliceosome was apparent. Later, highly significant networks of ubiquitin-mediated proteolysis and endoplasmic reticulum protein processing occurred. Finally, an enrichment of neuroinflammation factors appeared, together with profiles of bacterial invasion and MAPK signaling changes—while mitophagy had minor significance. Immunohistochemistry showed pronounced cellular response of Iba1-positive microglia and GFAP-positive astrocytes; brain lipidomics observed increases of ceramides as neuroinflammatory signs at old age. In a second approach, we assessed PINK1 deficiency in the presence of a stressor. Marked dysregulations of microbial defense factors Ifit3 and Rsad2 were consistently observed upon five analyses: (1) Pink1 −/− primary neurons in the first weeks after brain dissociation, (2) aged Pink1 −/− midbrain with transgenic A53T-alpha-synuclein overexpression, (3) human neuroblastoma cells with PINK1-knockdown and murine Pink1 −/− embryonal fibroblasts undergoing acute starvation, (4) triggering mitophagy in these cells with trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and (5) subjecting them to pathogenic RNA-analogue poly(I:C). The stress regulation of MAVS, RSAD2, DDX58, IFIT3, IFIT1, and LRRK2 was PINK1 dependent. Dysregulation of some innate immunity genes was also found in skin fibroblast cells from PARK6 patients. Conclusions Thus, an individual biomarker with expression correlating to progression was not identified. Instead, more advanced disease stages involved additional pathways. Hence, our results identify PINK1 deficiency as an early modulator of innate immunity in neurons, which precedes late stages of neuroinflammation during alpha-synuclein spreading.

Cyclic GMP-dependent protein kinase 1 (PKG1) mediates presynaptic nociceptive long-term potentiation (LTP) in the spinal cord and contributes to inflammatory pain in rodents but the present study revealed opposite effects in the context of neuropathic pain. We used a set of loss-of-function models for in vivo and in vitro studies to address this controversy: peripheral neuron specific deletion (SNS-PKG1-/-), inducible deletion in subsets of neurons (SLICK-PKG1-/-) and redox-dead PKG1 mutants. In contrast to inflammatory pain, SNS-PKG1-/- mice developed stronger neuropathic hyperalgesia associated with an impairment of nerve regeneration, suggesting specific repair functions of PKG1. Although PKG1 accumulated at the site of injury, its activity was lost in the proximal nerve due to a reduction of oxidation-dependent dimerization, which was a consequence of mitochondrial damage in injured axons. In vitro, PKG1 deficiency or its redox-insensitivity resulted in enhanced outgrowth and reduction of growth cone collapse in response to redox signals, which presented as oxidative hotspots in growing cones. At the molecular level, PKG1 deficiency caused a depletion of phosphorylated cofilin, which is essential for growth cone collapse and guidance. Hence, redox-mediated guidance required PKG1 and consequently, its deficiency in vivo resulted in defective repair and enhanced neuropathic pain after nerve injury. PKG1-dependent repair functions will outweigh its signaling functions in spinal nociceptive LTP, so that inhibition of PKG1 is no option for neuropathic pain. Keywords: Sensory neuron, Nerve regeneration, Pain, Growth cone, Signaling ROS, Cofilin, Redox

Parkinson's disease (PD) often manifests with prodromal pain and sensory losses whose etiologies are not well understood. Multiple genetic and toxicity-based rodent models of PD partly recapitulate the histopathology and motor function deficits. Although far less studied, there is some evidence that rodents, similar to humans, develop sensory manifestations of the disease, which may precede motor disturbances and help to elucidate the underlying mechanisms of PD-associated pain at the molecular and neuron circuit levels. The present Review summarizes nociception and other sensory functions in frequently used rodent PD models within the context of the complex phenotypes. In terms of mechanisms, it appears that the acute loss of dopaminergic neurons in systemic toxicity models (MPTP, rotenone) primarily causes nociceptive hyperexcitability, presumably owing to a loss of inhibitory control, whereas genetic models primarily result in a progressive loss of heat perception, reflecting sensory fiber neuropathies. At the molecular level, neither α-synuclein deposits alone nor failure of mitophagy alone appear to be strong enough to result in axonal or synaptic pathology of nociceptive neurons that manifest at the behavioral level, and peripheral sensory loss may mask central ‘pain’ in behavioral tests. Hence, allostatic combinations or additional challenges and novel behavioral assessments are needed to better evaluate PD-associated sensory neuropathies and pain in rodents.

Nucleoredoxin (NXN) is a redox regulator of Disheveled and thereby of WNT signaling. Deficiency in mice leads to cranial dysmorphisms and defects of heart, brain, and bone, suggesting defects of cell fate determination. We used shRNA-mediated knockdown of NXN in SH-SY5Y neuroblastoma cells to study its impact on neuronal cells. We expected that shNXN cells would easily succumb to redox stress, but there were no differences in viability on stimulation with hydrogen peroxide. Instead, the proliferation of naïve shNXN cells was increased with a higher rate of mitotic cells in cell cycle analyses. In addition, basal respiratory rates were higher, whereas the relative change in oxygen consumption upon mitochondrial stressors was similar to control cells. shNXN cells had an increased expression of redox-sensitive heat shock proteins, Hsc70/HSPA8 and HSP90, and autophagy markers suggested an increase in autophagosome formation upon stimulation with bafilomycin and higher flux under low dose rapamycin. A high rate of self-renewal, autophagy, and upregulation of redox-sensitive chaperones appears to be an attractive anti-aging combination if it were to occur in neurons in vivo for which SH-SY5Y cells are a model.

Injury of the sciatic nerve results in regulations of pro- and anti-oxidative enzymes at sites of nociceptive signaling including the injured nerve, dorsal root ganglia (DRGs), dorsal horn of the spinal cord, thalamus and somatosensory cortex (Valek et al., 2015) [1]. The present DiB paper shows immunohistochemistry of redoxins including peroxiredoxins (Prdx1–6), glutaredoxins (Glrx1, 2, 3, 5), thioredoxins (Txn1, 2) and thioredoxin reductases (Txnrd1, 2) in the DRGs, spinal cord and sciatic nerve and thalamus in naïve mice and 7 days after Spared sciatic Nerve Injury (SNI) in control mice (Hif1α-flfl) and in mice with a specific deletion of hypoxia inducible factor 1 alpha (SNS-HIF1α−/−) in DRG neurons. The sciatic nerves were immunostained for the respective redoxins and counterstained with hematoxylin. The redoxin immunoreactivity was quantified with ImageJ. For the DRGs and spinal cord the data show the quantitative assessment of the intensity of redoxin immunoreactivity transformed to rainbow pseudocolors. In addition, some redoxin examples of the ipsi and contralateral dorsal and ventral horns of the lumbar spinal cord and some redoxin examples of the thalamus are presented.