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As neurons establish extensive connections throughout the central nervous system, the transport of cargo along the microtubule network of the axon is crucial for differentiation and homeostasis. Specifically, building blocks such as membrane and cytoskeletal components, organelles, transmembrane receptors, adhesion molecules, and peptide neurotransmitters all require proper transport to the presynaptic compartment. Here, we identify a novel complex regulating vesicular endoplasmic reticulum transport in neurites, composed of CLN6: an ER-associated protein of relatively unknown function implicated in CLN6-Batten disease; CRMP2: a tubulin binding protein important in regulating neurite microtubule dynamics; and KLC4: a classic transport motor protein. We show that this “CCK” complex allows ER-derived vesicles to migrate to the distal end of the axon, aiding in proper neurite outgrowth and arborization. In the absence of CLN6, the CCK complex does not function effectively, leading to reduced vesicular transport, stunted neurite outgrowth, and deficits in CRMP2 binding to other protein partners. Treatment with a CRMP2 modulating compound, lanthionine ketimine ester, partially restores these deficits in CLN6-deficient mouse neurons, indicating that stabilization of CRMP2 interacting partners may prove beneficial in lieu of complete restoration of the CCK complex. Taken together, these findings reveal a novel mechanism of ER-derived vesicle transport in the axon and provide new insights into therapeutic targets for neurodegenerative disease.

Lanthionine synthase C-like protein-1 (LanCL1) is a glutathione (GSH)-binding protein of uncertain function, widely expressed in mammalian cells. Recent data suggests that LanCL1 has glutathione S-transferase (GST)-like activity, while other reports claim that LanCL1 suppresses mitogen-activated kinase (MAPK) phosphorylation. In the present study, recombinant human LanCL1 had less than 10% the specific activity of GST. When CRISPR-Cas9 was used to stably ablate LanCL1 from HeLa cells, the resulting line was sensitized to H2O2 toxicity. [GSH], [GSSG], [GSH]/[GSSG] and GST activity were unaltered by LanCL1 knockout but glutathione reductase and glutathione peroxidase activities were significantly elevated. LanCL1-KO cells did not differ in basal or H2O2-induced p38-MAPK, ERK p42/p44 or JNK phosphorylation; however, MAPK-targeted transcription factor regulators c-Jun and IκBα were significantly decreased. Because c-Jun and IκBα levels are ubiquitin regulated, experiments addressed the hypothesis that LanCL1 affects ubiquitination dynamics. In the presence of the 26S proteasome inhibitor bortezomib, ubiquitinated proteins accumulated faster in LanCL1-KO cells, suggesting that LanCL1 positively regulates deubiquitination. The activity of ubiquitin C-terminal hydrolase (UCH), a major deubiquitinase (DUB) subclass, was significantly decreased in LanCL1-KO cells while protein levels of A20/TNFAIP3, USP9X and USP10 DUBs were significantly reduced. UCH activity in HeLa cell lysates was lost upon treatment with H2O2 and significantly recovered by addition of recombinant LanCL1 plus GSH. Taken together these data suggest that LanCL1 likely does not act as a GST-like enzyme in vivo, but rather modulates ubiquitin-dependent cell signaling pathways through positive regulation of redox-sensitive DUBs.

Okadaic acid (OKA) is a protein phosphatase 2A inhibitor that is used to induce neurodegeneration and study disease states such as Alzheimer's disease (AD). Lanthionine ketimine-5-ethyl ester (LKE) is a bioavailable derivative of the naturally occurring brain sulfur metabolite, lanthionine ketimine (LK). In previously conducted studies, LKE exhibited neuroprotective and neurotrophic properties in murine models but its mechanism of action remains to be clarified. In this study, a recently established zebrafish OKA-induced AD model was utilized to further elucidate the neuroprotective and neurotrophic properties of LKE in the context of an AD-like condition. The fish were divided into 3 groups containing 8 fish per group. Group #1 = negative control, Group #2 = 100 nM OKA, Group #3 = 100 nM OKA +500 μM LKE. OKA caused severe cognitive impairments in the zebrafish, but concomitant treatment with LKE protected against cognitive impairments. Further, LKE significantly and substantially reduced the number of apoptotic brain cells, increased brain-derived neurotrophic factor (BDNF), and increased phospho-activation of the pro-survival factors pAkt (Ser 473) and pCREB (Ser133). These findings clarify the neuroprotective and neurotrophic effects of LKE by highlighting particular survival pathways that are bolstered by the experimental therapeutic LKE.

The multiple-step, one-pot procedure for a series of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates, analogues of the natural, sulfur amino acid metabolite lanthionine ketimine (LK), its 5-ethyl ester (LKE) and 2-substituted LKEs is described. Initiating the synthesis with the Michaelis-Arbuzov preparation of α-ketophosphonates allows for a wide range of functional variation at the 2-position of the products. Nine new compounds were synthesized with overall yields range from 40 to 62%. In addition, the newly prepared 2-isopropyl-LK-P, 2-n-hexyl-LKE-P and 2-ethyl-LKE were shown to stimulate autophagy in cultured cells better than that of the parent compound, LKE.

We previously showed that treatment with lanthionine ketimine ethyl ester (LKE) reduced disease severity and axonal damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis and increased neuronal maturation and survival in vitro. A major target of LKE is collapsin response mediator protein 2 (CRMP2), suggesting this protein may mediate LKE actions. We now show that conditional knockout of CRMP2 from neurons using a CamK2a promoter to drive Cre recombinase expression reduces disease severity in the myelin oligodendrocyte glycoprotein (MOG)35–55 EAE model, associated with decreased spinal cord axonal damage, and less glial activation in the cerebellum, but not the spinal cord. Immunohistochemical staining and quantitative polymerase chain reaction show CRMP2 depletion from descending motor neurons in the motor cortex, but not from spinal cord neurons, suggesting that the benefits of CRMP2 depletion on EAE may stem from effects on upper motor neurons. In addition, mice in which CRMP2 S522 phosphorylation was prevented by substitution for an alanine residue also showed reduced EAE severity. These results show that modification of CRMP2 expression and phosphorylation can influence the course of EAE and suggests that treatment with CRMP2 modulators such as LKE act in part by reducing CRMP2 S522 phosphorylation.

Collapsin response mediator protein 2 (CRMP-2) is a neuronal protein involved in axonal pathfinding. Intense research is focusing on its role in various neurological diseases. Despite a wealth of studies, not much is known about the molecular mechanisms of CRMP-2 function in vivo. The detailed structure-function relationships of CRMP-2 have also largely remained unknown, in part due to the fact that the available crystal structures lack the C-terminal tail, which is known to be a target for many post-translational modifications and protein interactions. Although CRMP-2, and other CRMPs, belong to the dihydropyrimidinase family, they have lost the enzymatic active site. Drug candidates for CRMP-2-related processes have come up during the recent years, but no reports of CRMP-2 complexes with small molecules have emerged. Here, CRMP-2 was studied at 1.25-Å resolution using X-ray crystallography. In addition, ligands were docked into the homotetrameric structure, and the C-terminal tail of CRMP-2 was produced recombinantly and analyzed. We have obtained the human CRMP-2 crystal structure at atomic resolution and could identify small-molecule binding pockets in the protein. Structures obtained in different crystal forms highlight flexible regions near possible ligand-binding pockets. We also used the CRMP-2 structure to analyze known or suggested post-translational modifications at the 3D structural level. The high-resolution CRMP-2 structure was also used for docking experiments with the sulfur amino acid metabolite lanthionine ketimine and its ester. We show that the C-terminal tail is intrinsically disordered, but it has conserved segments that may act as interaction sites. Our data provide the most accurate structural data on CRMPs to date and will be useful in further computational and experimental studies on CRMP-2, its function, and its binding to small-molecule ligands.

Alzheimer's disease (AD) has long been viewed as a pathology that must be caused either by aberrant amyloid-β protein precursor (AβPP) processing, dysfunctional tau protein processing, or a combination of these two factors. This is a reasonable assumption because amyloid-β peptide (Aβ) accumulation and tau hyperphosphorylation are the defining histological features in AD, and because AβPP and tau mutations can cause AD in humans or AD-like features in animal models. Nonetheless, other protein players are emerging that one can argue are significant etiological players in subsets of AD and potentially novel, druggable targets. In particular, the microtubule-associated protein CRMP2 (collapsin response mediator protein-2) bears striking analogies to tau and is similarly relevant to AD. Like tau, CRMP2 dynamically regulates microtubule stability; it is acted upon by the same kinases; collects similarly in neurofibrillary tangles (NFTs); and when sequestered in NFTs, complexes with critical synapse-stabilizing factors. Additionally, CRMP2 is becoming recognized as an important adaptor protein involved in vesicle trafficking, amyloidogenesis and autophagy, in ways that tau is not. This review systematically compares the biology of CRMP2 to that of tau in the context of AD and explores the hypothesis that CRMP2 is an etiologically significant protein in AD and participates in pathways that can be rationally engaged for therapeutic benefit.

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Autophagy and redox biochemistry are two major sub disciplines of cell biology which are both coming to be appreciated for their paramount importance in the etiology of neurodegenerative diseases including Alzheimer's disease (AD). Thus far, however, there has been relatively little exploration of the interface between autophagy and redox biology. Autophagy normally recycles macro-molecular aggregates produced through oxidative-stress mediated pathways, and also may reduce the mitochondrial production of reactive oxygen species through recycling of old and damaged mitochondria. Conversely, dysfunction in autophagy initiation, progression or clearance is evidenced to increase aggregation-prone proteins in neural and extraneural tissues. Redox mechanisms of autophagy regulation have been documented at the level of cross-talk between the Nrf2/Keap1 oxidant and electrophilic defense pathway and p62/sequestosome-1 (SQSTM1)-associated autophagy, at least in extraneural tissue; but other mechanisms of redox autophagy regulation doubtless remain to be discovered and the relevance of such processes to maintenance of neural homeostasis remains to be determined. This review summarizes current knowledge regarding the relationship of redox signaling, autophagy control, and oxidative stress as these phenomena relate to neurodegenerative disease. AD is specifically addressed as an example of the theme and as a promising indication for new therapies that act through engagement of autophagy pathways. To exemplify one such novel therapeutic entity, data is presented that the antioxidant and neurotrophic agent lanthionine ketimine-ethyl ester (LKE) affects autophagy pathway proteins including beclin-1 in the 3xTg-AD model of Alzheimer's disease where the compound has been shown to reduce pathological features and cognitive dysfunction.

Oxidative stress triggers and exacerbates neurodegeneration in Alzheimer's disease (AD). Various antioxidants reduce oxidative stress, but these agents have little efficacy due to poor blood-brain barrier (BBB) permeability. Additionally, single-modal antioxidants are easily overwhelmed by global oxidative stress. Activating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream antioxidant system are considered very effective for reducing global oxidative stress. Thus far, only a few BBB-permeable agents activate the Nrf2-dependent antioxidant system. Here, we discovered a BBB-bypassing Nrf2-activating polysaccharide that may attenuate AD pathogenesis. Mini-GAGR, a 0.7-kDa cleavage product of low-acyl gellan gum, increased the levels and activities of Nrf2-dependent antioxidant enzymes, decreased reactive oxygen species (ROS) under oxidative stress in mouse cortical neurons, and robustly protected mitochondria from oxidative insults. Moreover, mini-GAGR increased the nuclear localization and transcriptional activity of Nrf2 similarly to known Nrf2 activators. Mechanistically, mini-GAGR increased the dissociation of Nrf2 from its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), and induced phosphorylation and nuclear translocation of Nrf2 in a protein kinase C (PKC)- and fibroblast growth factor receptor (FGFR1)-dependent manner. Finally, 20-day intranasal treatment of 3xTg-AD mice with 100 nmol of mini-GAGR increased nuclear p-Nrf2 and growth-associated protein 43 (GAP43) levels in hippocampal neurons, reduced p-tau and β-amyloid (Aβ) peptide-stained neurons, and improved memory. The BBB-bypassing Nrf2-activating polysaccharide reported here may be effective in reducing oxidative stress and neurodegeneration in AD.

The oxidation of omega-3 fatty acids, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and alpha-linolenic acid (ALA), was studied by MS and tandem mass spectrometry (MS/MS) using a custom-designed DESI source, which was coupled to an ion trap (IT)-mass spectrometer. DESI-MS showed that oxidation due to aging occurred most readily by peroxidations and keto formations of the omega-3 fatty acids, which are the expected oxidation pathways for these molecules. The oxidized products detected were compared between DESI-MS and ESI-MS after a three-week period of sample aging at the room temperature and ambient conditions. The mass spectra of the omega-3 fatty acids obtained by these two techniques in negative ion mode were similar, while oxidation of omega-3 fatty acids was observable with DESI-MS in positive ion mode only after prolonged aging of the samples. The peroxidation products of fatty acids, similar to those observed by DESI and ESI-MS, were also detected by MALDI-MS. Additionally, a commercial dog kibble was analyzed by DESI-MS. DHA, EPA, and some possible oxidation products were identified from the surface of this food sample using DESI-MS and MS/MS. The results demonstrate that DESI-MS can be used efficiently in negative ion mode to monitor oxidation of fatty acids due to their aging both in pure forms and on the surfaces of solid food samples.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by progressive distal axonopathy that precedes actual motor neuron death. Triggers for neuromuscular junction degeneration remain to be determined, but the axon repulsion factor semaphorin 3A (Sema3A), which is derived from terminal Schwann cells, is a plausible candidate. This study examines the hypothesis that Sema3A signaling through its motor neuron neuropilin-1 (NRP1) receptor triggers distal axonopathy and muscle denervation in the SOD1G93A mouse model of ALS. Neuropilin-1 was found to be expressed in axonal terminals at the mouse neuromuscular junction in vivo and in NSC-34 motor neuron–like cells in vitro. In differentiated NSC-34 cells, an anti-NRP1A antibody that selectively blocks Sema3A binding to NRP1 prevented Sema3A-induced growth cone collapse. Furthermore, intraperitoneal injections of anti-NRP1A antibody administered twice weekly from age 40 days significantly delayed and even temporarily reversed motor functional decline while prolonging the life span of SOD1G93A mice. Histologic evaluation at 90 and 125 days revealed that anti-NRP1A antibody reduced neuromuscular junction denervation and attenuated pathologic alterations in ventral roots at late-stage disease. These data suggest that peripheral NRP1A signaling is involved in the pathobiology of this ALS model and that antagonizing Sema3A/NRP1 binding or downstream signals could have implications for the treatment of ALS.

Lanthionine ketimine ([LK] 3,4-dihydro-2H-1,4-thiazine-3,5-dicarboxylic acid) is the archetype for a family of naturally occurring brain sulfur amino acid metabolites, the physiologic function of which is unknown. Lanthionine ketimine and its synthetic derivatives have recently demonstrated neurotrophic, neuroprotective, and antineuroinflammatory properties in vitro through a proposed mechanism involving the microtubule-associated protein collapsin response mediator protein 2. Therefore, studies were undertaken to test the effects of a bioavailable LK ester in the 3 × Tg-AD mouse model of Alzheimer disease. Lanthionine ketimine ester treatment substantially diminished cognitive decline and brain amyloid-β (Aβ) peptide deposition and phospho-Tau accumulation in 3 × Tg-AD mice and also reduced the density of Iba1-positive microglia. Furthermore, LK ester treatment altered collapsin response mediator protein 2 phosphorylation. These findings suggest that LK may not be a metabolic waste but rather a purposeful neurochemical, the synthetic derivatives of which constitute a new class of experimental therapeutics for Alzheimer disease and related entities.

Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time in order to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively-generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this method article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO).

Context: Quantitative changes of salivary proteins due to acute stress were detected. Objective: To explore protein markers of stress in saliva of eight medical residents who performed emergency medicine simulations. Materials and methods: Saliva was collected before the simulations, after the simulations, and following morning upon waking. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), identified by mass spectrometry (MS), and relatively quantified by densitometry. Results: Salivary alpha-amylase and S–type cystatins significantly increased, while the ∼26 kDa and low-molecular weight (MW) (Discussion and conclusion: Alpha-amylase and cystatins are potential salivary markers of acute stress, but further validation should be performed using larger sample populations.

CRMP2, also known as DPYSL2/DRP2, Unc-33, Ulip or TUC2, is a cytosolic phosphoprotein that mediates axon/dendrite specification and axonal growth. Mapping the CRMP2 interactome has revealed previously unappreciated functions subserved by this protein. Together with its canonical roles in neurite growth and retraction and kinesin-dependent axonal transport, it is now known that CRMP2 interacts with numerous binding partners to affect microtubule dynamics; protein endocytosis and vesicular cycling, synaptic assembly, calcium channel regulation and neurotransmitter release. CRMP2 signaling is regulated by post-translational modifications, including glycosylation, oxidation, proteolysis and phosphorylation; the latter being a fulcrum of CRMP2 functions. Here, the putative roles of CRMP2 in a panoply of neurodegenerative, sensory and motor neuron, and central disorders are discussed and evidence is presented for therapeutic strategies targeting CRMP2 functions.

The objective of this study was to determine whether exposing rats to ozone would result in loss of antioxidants from plasma and bronchoalveolar lavage fluid (BALF). Additional goals were to compare analyses of the same antioxidant concentration between different laboratories, to investigate which methods have the sensitivity to detect decreased levels of antioxidants, and to identify a reliable measure of oxidative stress in ozone-exposed rats. Male Fisher rats were exposed to either 2.0 ppm or 5.0 ppm ozone inhalation for 2 h. Blood plasma and BALF samples were collected 2 h, 7 h, and 16 h after the exposure. It was found that ascorbic acid in plasma collected from rats after the higher dose of ozone was lower at 2h but not later. BALF concentrations of ascorbic acid were decreased at both 2 and 7 h post-exposure. Tocopherols (α-, δ-, γ-), 5-nitro-γ-tocopherol, tocol, glutathione (GSH/GSSG) and cysteine (Cys/CySS) were not further decreased, regardless of the doses and post-exposure time points used for sample collection. Uric acid was significantly increased by the low dose at 2h and the high dose at the 7 h point, probably due to the accumulation of blood plasma in the lung from ozone-increased alveolar capillary permeability. We concluded that measurements of antioxidants in plasma are not sensitive biomarkers for oxidative damage induced by the ozone and is not a useful choice for the assessment of oxidative damage by ozone in vivo.

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.

Lanthionine (Lan), the thioether analog of cystine, is a natural but nonproteogenic amino acid thought to form naturally in mammals through promiscuous reactivity of the transsulfuration enzyme cystathionine-beta-synthase (CbetaS). Lanthionine exists at appreciable concentrations in mammalian brain, where it undergoes aminotransferase conversion to yield an unusual cyclic thioether, lanthionine ketimine (LK; 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid). Recently, LK was discovered to possess neuroprotective, neuritigenic and anti-inflammatory activities. Moreover, both LK and the ubiquitous redox regulator glutathione (gamma-glutamyl-cysteine-glycine) bind to mammalian lanthionine synthetase-like protein-1 (LanCL1) protein which, along with its homolog LanCL2, has been associated with important physiological processes including signal transduction and insulin sensitization. These findings begin to suggest that Lan and its downstream metabolites may be physiologically important substances rather than mere metabolic waste. This review summarizes the current state of knowledge about lanthionyl metabolites with emphasis on their possible relationships to LanCL1/2 proteins and glutathione. The potential significance of lanthionines in paracrine signaling is discussed with reference to opportunities for utilizing bioavailable pro-drug derivatives of these compounds as novel pharmacophores.

The concept of neuroinflammation has evolved over the past two decades from an initially controversial viewpoint to its present status as a generally accepted idea whose mechanisms and consequences are still actively under research and debate, particularly with regard to Alzheimer’s disease (AD). This review summarizes the current status of neuroinflammation research as it specifically relates to AD. Neuroinflammation is discussed mechanistically with emphasis on the role of redox signal transduction linked to the activation of central nervous system-relevant innate immune pathways. Redox signaling is presented both as a causal factor and a consequence of sustained neuroinflammation. Functional relationships are discussed that connect distinct neuroinflammatory components such as cytokines, eicosanoids, classic AD pathology (amyloid plaques and neurofibrillary tangles), and the recently emergent notion of “damage-associated molecular patterns”. The interaction of these paracrine factors likely can produce positive as well as negative effects on the AD brain, ranging from plaque clearance by microglia in the short term to glial dysfunction and neuronal compromise if the neuroinflammation is chronically sustained and unmitigated. Recent disappointments in AD clinical trials of anti-inflammatory drugs are discussed with reference to possible explanations and potential avenues for future pharmacological approaches to the disease.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which is currently untreatable. Inflammation plays a major role in the pathogenesis of motor neuron death in ALS. Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and Fas ligand (FasL) are amongst the most important mediators of neuro-inflammation. We have previously demonstrated that elevation of these pro-inflammatory cytokines occurs in both ALS transgenic mice and in human ALS postmortem spinal cord tissues. Lenalidomide is a potent immunomodulatory agent, with the ability to down-regulate pro-inflammatory cytokines and up-regulate anti-inflammatory cytokines. We previously reported the neuroprotective effects of lenalidomide, when treatment was started 2 months prior to onset of disease in the G93A SOD1 transgenic mouse model of ALS. Since in ALS patients, treatment can only begin after the appearance of symptoms, we sought to determine the efficacy of lenalidomide administration starting at symptom onset in the G93A SOD1 mice. We found that lenalidomide treatment extended the survival interval from the age of onset by 18.3 days ( approximately 45%). Additionally, lenalidomide treatment improved rotarod performance, reduced weight loss, and attenuated neuronal cell death in the lumbar spinal cord. Qualitative histological analysis showed that lenalidomide treatment modestly reduced the expression of the proinflammatory cytokines Fas Ligand, IL-1beta, TNF-alpha and CD40 ligand. RNA protection Assay (RPA) on a pre-selected panel of cytokines showed that proinflammatory cytokines were reduced and anti-inflammatory cytokines were up-regulated. These data encourage further clinical evaluation of lenalidomide as therapeutic strategy to block or slow disease progression in human ALS patients.

Eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, yet its biochemical functions remain elusive. We report the crystal structures of human LanCL1, both free of and complexed with glutathione, revealing glutathione binding to a zinc ion at the putative active site formed by conserved GxxG motifs. We also demonstrate by in vitro affinity analysis that LanCL1 binds specifically to the SH3 domain of a signaling protein, Eps8. Importantly, expression of LanCL1 mutants defective in Eps8 interaction inhibits nerve growth factor (NGF)-induced neurite outgrowth, providing evidence for the biological significance of this novel interaction in cellular signaling and differentiation.

Human life expectancy and welfare has decreased because of the increase in environmental stressors in the air. An environmental stressor is a natural or human-made component present in our environment that upon reaching an organic system produces a coordinated response. This response usually involves a modification of the metabolism and physiology of the system. Inhaled environmental stressors damage the airways and lung parenchyma, producing irritation, recruitment of inflammatory cells, and oxidative modification of biomolecules. Oxidatively modified biomolecules, their degradation products, and adducts with other biomolecules can reach the systemic circulation, and when found in higher concentrations than normal they are considered to be biomarkers of systemic oxidative stress and inflammation. We classify them as metabolic stressors because they are not inert compounds; indeed, they amplify the inflammatory response by inducing inflammation in the lung and other organs. Thus the lung is not only the target for environmental stressors, but it is also the source of a number of metabolic stressors that can induce and worsen pre-existing chronic inflammation. Metabolic stressors produced in the lung have a number of effects in tissues other than the lung, such as the brain, and they can also abrogate the mechanisms of immunotolerance. In this review, we discuss recent published evidence that suggests that inflammation in the lung is an important connection between air pollution and chronic inflammatory diseases such as autoimmunity and neurodegeneration, and we highlight the critical role of metabolic stressors produced in the lung. The understanding of this relationship between inhaled environmental pollutants and systemic inflammation will help us to: (1) understand the molecular mechanism of environment-associated diseases, and (2) find new biomarkers that will help us prevent the exposure of susceptible individuals and/or design novel therapies.

The p38 mitogen-activated protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimer's disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age-matched normal brain. Intense phospho-p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle-bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF-positive tau did not cross-react with the phospho-p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.

A major genetic risk factor for sporadic Alzheimer’s disease is inheritance of the epsilon-4 allele of apolipoprotein E (Corder et al., 1993; Poirier et al., 1993a; Rebeck et al., 1993; Saunders et al., 1993; Crutcher, 2004). ApoE-4 is also associated with an earlier age of onset of the disease (Corder et al., 1993) and an increased amyloid plaque burden (Mortimer et al., 1991; Schemechel et al., 1993; Sadowski et al., 2004). There is significant evidence suggesting that the interactions between apoE and Aβ are critical for the development of Aβ deposits (Strittmatter et al., 1993). Apolipoprotein-E (ApoE) is a 34-kDa lipoprotein synthesized within the CNS that regulates plasma lipid transport and clearance. It is involved in nerve regeneration after injury (Mahley, 1988; Boyles et al., 1989; LeBlanc and Poduslo, 1990). Traumatic brain injury (e.g., hemorrhagic stroke, head injury), wherein neurons are exposed to high thrombin levels, is a significant risk factor for AD (Mayeaux et al., 1995). Moreover, this risk increases several times in individuals possessing the ApoE-4 allele relative to people inheriting ApoE-2 and ApoE-3 alleles (Mortimer et al., 1991; Mayeaux et al., 1995; Teasdale et al., 1997). Also, AD risk is significantly greater in ApoE-4 carriers, who have experienced head trauma than in those without a history of head trauma (Kutner et al., 2000). The E4 iso-form is also associated with neurologic deficits in the survivors of traumatic brain injury (Wisniewski et al., 1994; Teasdale et al., 1997; Friedman, 1999) and is a negative risk factor for recovery from head trauma (Friedman et al., 1999; Graham et al., 1999, 2000; Kutner et al., 2000). Aged football players possessing ApoE-4 iso-form have shown significantly lower cognitive performance than E2- or E3-carriers (Kutner et al., 2000). These observations suggest a significant interaction between traumatic brain injury and ApoE4 allele. We observed previously that chronic exposure to thrombin, which is released at a high concentration after hemorrhagic stroke or traumatic brain injury, produces significant cognitive deficits that can be prevented by direct and specific inhibition of thrombin by hirudin (Mhatre et al., 2004) or thrombin receptor (PAR-1) antagonist (unpublished observations). Thrombin-induced cognitive deficits were associated with ventricular enlargement, increased number of reactive glial fibrillary acidic protein-positive astrocytes and TUNEL-positive cells, and alterations in the phosphorylation state of cytoskeletal proteins suggesting neuronal injury (Mhatre et al., 2004). Emmerling et al. (2000) observed previously that the levels of Aβ1–42 increase in a positive correlation with apolipoprotein-E levels in the cerebrospinal fluid of patients after severe brain injury. Despite the positive correlation between apolipoprotein-E and Aβ1–42 levels observed in these patients, it is not yet clear whether the increase in apolipoprotein-E levels after brain injury in ApoE4-carriers contributes to Aβ deposition. The present study is designed to determine whether chronic exposure to thrombin as observed after brain injury alters apolipoprotein-E levels and whether such alterations in apolipoprotein-E levels in ApoE4-carriers contribute to β-amyloid deposition.

Research and key discoveries in the field of amyotrophic lateral sclerosis (ALS) have exponentially increased since the announcement in 1993 of the first ALS-causing mutations in the gene for the well-studied antioxidant enzyme Cu,Zn superoxide dismutase (SOD1). The etiology of sporadic ALS largely remains unknown and the mechanisms of motor neuron degeneration are still being investigated. The only FDA drug approved for the treatment of ALS is riluzole with only modest benefit to patients, but multiple drugs are currently in the development pipeline and in human ALS clinical trials. In this special issue, K. Venkova-Hristova et al. thoroughly reviewed studies of experimental therapeutics in animal models of ALS, including specific examples of those that proceeded into human clinical trials. Since none of the ALS human clinical trials succeeded despite positive results in animal models, the question of “why” has been on everyone's mind, with efforts to develop superior alternatives. This review discusses the potential reasons for the universal failure of preclinical successes to translate into positive clinical outcomes. It has become obvious that the lack of understanding of the precise mechanisms of motor neuron degeneration presents a major obstacle in the development of effective therapy for ALS. This review discusses the details of several major pathogenic pathways in ALS and the efforts of various groups to block one toxic pathway at a time, ranging from oxidative stress to protein aggregation. The authors discuss the pros and cons of ALS models and propose simultaneous targeting of multiple pathways as a more efficient strategy, due to the multifactorial nature of ALS pathology.

The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the second-order reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated beta-amyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.

Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O 2 to hydrogen peroxide (H 2 O 2 ) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H 2 O 2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O 2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H 2 O 2 generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H 2 O 2 flux was accelerated by rotenone. α-Phenyl-N-tert-butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex 1-stimulated H 2 O 2 flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H 2 O 2 synthesis, possessing an electron leak site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H 2 O 2 production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.

Scientific appreciation for the subtlety of brain sulfur chemistry has lagged, despite understanding that the brain must maintain high glutathione (GSH) to protect against oxidative stress in tissue that has both a high rate of oxidative respiration and a high content of oxidation-prone polyunsaturated fatty acids. In fact, the brain was long thought to lack a complete transsulfuration pathway (TSP) for cysteine synthesis. It is now clear that not only does the brain possess a functional TSP, but brain TSP enzymes catalyze a rich array of alternative reactions that generate novel species including the gasotransmitter hydrogen sulfide (H2S) and the atypical amino acid lanthionine (Lan). Moreover, TSP intermediates can be converted to unusual cyclic ketimines via transamination. Cell-penetrating derivatives of one such compound, lanthionine ketimine (LK), have potent antioxidant, neuroprotective, neurotrophic, and antineuroinflammatory actions and mitigate diverse neurodegenerative conditions in preclinical rodent models. This review will explore the source and function of alternative TSP products, and lanthionine-derived metabolites in particular. The known biological origins of lanthionine and its ketimine metabolite will be described in detail and placed in context with recent discoveries of a GSH- and LK-binding brain protein called LanCL1 that is proving essential for neuronal antioxidant defense; and a related LanCL2 homolog now implicated in immune sensing and cell fate determinations. The review will explore possible endogenous functions of lanthionine metabolites and will discuss the therapeutic potential of lanthionine ketimine derivatives for mitigating diverse neurological conditions including Alzheimer׳s disease, stroke, motor neuron disease, and glioma.

Free radicals are compounds with an unpaired electron capable of independent existence. These highly reactive species have been implicated in many disease states and can react with cell membranes, lipids, proteins, and DNA. When an oxygen radical reacts with DNA, base damage, cross-linking (e.g., DNA-DNA or DNA-protein), or DNA backbone damage (e.g., single- or double-strand breaks) can occur and often result in cell death. The field of oxidative metabolism as it relates to DNA damage has grown tremendously, with more DNA adducts being identified as biomarkers. These biomarkers are indicative of DNA damage. Measurement of these biomarkers has proved to be a challenge because of their relatively low occurrence (1 per 10(5)-10(6) bases). Methodologies for the measurement of DNA damage include thin-layer chromatography, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, DNA sequencing, high-performance liquid chromatography (HPLC)-ultraviolet, and HPLC-ECD. HPLC-ECD (electrochemical detection) is a powerful technique that is both sensitive and selective. However, HPLC-ECD is generally not amenable to gradient analyses, so its utility is restricted. In addition, many of the bases and nucleosides are not electrochemically active. Gradient HPLC separation coupled to both a coulometric electrochemical array detector and an ultraviolet detector overcomes these limitations. Presented here is a gradient HPLC method that measures a wide variety of nucleosides, bases, and hydroxylated adducts using the inherent stability, sensitivity, and wide dynamic range of a coulometric electrochemical array detector and the universal detection qualities of an ultraviolet detector. Linear ranges, limits of detection, and detailed methods development are presented.

Pheochromocytoma originates from chromaffin cells in the adrenal medulla and sympathetic paraganglia. 36-53% of pheochromocytoma becomes malignant and, thereafter, resistant to conventional treatments. Pheochromocytoma also causes hyper-secretion of catecholamines that cause severe hypertension. We found that an antidepressant, tianeptine, interfered with normal life cycle of pheochromocytoma cells at its clinical doses. Treatment with tianeptine caused microtubule bundling and specific degradation of cytoplasmic dynein, a retrograde microtubule motor that mediates various microtubule-dependent processes during interphase and mitosis, in the rat pheochromocytoma PC12 cells. Tianeptine also increased the levels of pro-apoptotic proteins, slowed cell cycle progression, and increased apoptosis in PC12 cells. Importantly, tianeptine treatment decreased high K(+)-stimulated secretion of norepinephrine and chromogranin A in PC12 cells and of epinephrine in the mouse pheochromocytoma MPC cells. Our study demonstrates, for the first time, that tianeptine interferes with normal life cycle of pheochromocytoma cells.

Please consider the following changes in Table 1. Table 1 Mendelian and non-Mendelian loci known to cause FALS or confer risk for SALS. Information in this table reproduces content in Tables 7 and 8 from Lill and Bertram, 2011 ([2], and databases therein). Polymorphisms in the VEGF promoter ...

The possibility of free radical reactions occurring in biological processes led to the development and employment of novel methods and techniques focused on determining their existence and importance in normal and pathological conditions. For this reason the use of Nitrones for spin trapping free radicals came into widespread use in the 1970s and 1980s when surprisingly the first evidence of their potent biological properties was first noted. Since then wide-spread exploration and demonstration of the potent biological properties of phenyl-tert-butylnitrone (PBN) and derivatives were shown in preclinical models of septic shock and then in experimental stroke. The most extensive commercial effort done in order to capitalize on the potent properties of the PBN-Nitrones was for acute ischemic stroke. This occurred during the 1993–2006 time interval where the 2,4-disulfonyl-phenyl PBN derivative, called NXY-059 in the stroke studies, was shown to be safe in humans and was taken all the way through clinical phase 3 trials and was deemed to be ineffective. As summarized in this review because of its excellent human safety profile 2,4-disulfonyl-phenyl PBN, now called OKN-007 in the cancer studies, was tested as an anti-cancer agent in several preclinical glioma models and shown to be very effective. Based on these studies this compound is now scheduled to enter into early clinical trials for astrocytoma/glioblastoma multiform this year. The potential use of OKN-007 in combination with neurotropic compounds such as the lanthionine ketamine esters (LKE) is discussed for glioblastoma multiform as well as for various other indications leading to dementia such as aging, septic shock, and malaria infections. There is much more research and development activity on-going for various indications with the nitrones alone or in combination with other active compounds as briefly noted in this review.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that cannot be slowed substantially using any currently-available clinical tools. Through decades of studying sporadic and familial ALS (SALS and FALS), researchers are coming to understand ALS as a complex syndrome with diverse genetic and environmental etiologies. It is know appreciated that motor neuron degeneration in ALS requires active (gain of function) and passive (loss of function) events to occur in non-neuronal cells, especially astrocytes and microglia. These neuroinflammatory processes produce paracrine factors that detrimentally affect motor neurons, precipitating protein aggregation and compromising cytoskeletal integrity. The result is a loss of neuronal homeostasis and progressive die-back of motor axons culminating in death of the afflicted motor neurons. This review will discuss experimental therapeutics that have been tested in murine ALS models, with an emphasis on those that have progressed to human clinical trials. Reasons will be considered for the frequent failure of preclinical successes to translate into positive clinical outcomes. Finally, this review will explore current trends in experimental therapeutics for ALS with emphasis on the emerging interest in axon guidance signaling pathways as novel targets for pharmacological support of neural cytoskeletal structure and function in order to slow ALS.

Over the past two decades, it has become increasingly apparent that Alzheimer’s disease neuropathology is characterized by activated microglia (brain resident macrophages) as well as the classic features of amyloid plaques and neurofibrillary tangles. The intricacy of microglial biology has also become apparent, leading to a heightened research interest in this particular cell type. Over the years a number of different microglial cell culturing techniques have been developed to study either primary mammalian microglia, or immortalized cell lines. Each microglial system has advantages and disadvantages and should be selected for its appropriateness in a particular research context. This review summarizes several of the most common microglial cell culture systems currently being employed in Alzheimer’s research including primary microglia; BV2 and N9 retroviral immortalized microglia; human immortalized microglia (HMO6); and spontaneously immortalized rodent microglial lines (EOC lines and HAPI cells). Particularities of cell culture requirements and characteristics of microglial behavior, especially in response to applied inflammogen stimuli, are compared and discussed across these cell types.

Lanthionines are novel neurotrophic and neuroprotective small molecules that show promise for the treatment of neurodegenerative diseases. In particular, a recently developed, cell permeable lanthionine derivative known as LKE (lanthionine ketimine 5-ethyl ester) promotes neurite growth at low nanomolar concentrations. LKE also has neuroprotective, anti-apoptotic, and anti-inflammatory properties. Its therapeutic potential in cerebral ischemia and its mechanisms of neurotrophic action remain to be fully elucidated. Here, we hypothesize that the neuroprotective actions of LKE could result from induction or modulation of CRMP2. We found that treating primary cultured mouse neurons with LKE provided significant protection against t-butyl hydroperoxide-induced neuronal death possibly through CRMP2 upregulation. Similarly, in-vivo studies showed that LKE pre and/or post-treatment protects mice against permanent distal middle cerebral artery occlusion (p-MCAO) as evidenced by lower stroke lesions and improved functional outcomes in terms of rotarod, grip strength and neurologic deficit scores in treated groups. Protein expression levels of CRMP2 were higher in brain cortices of LKE pretreated mice, suggesting that LKE’s neuroprotective activity may be CRMP2 dependent. Lower activity of cleaved PARP and higher activity of SERT1 was also observed in LKE treated group suggesting its anti-apoptotic properties. Our results suggest that LKE has potential as a therapeutic intervention in cerebral ischemia and that part of its protective mechanism may be attributed in- part to CRMP2 mediated action and PARP-1/SIRT-1 modulation.

There is ample mounting evidence that reactive oxidant species are exacerbated in inflammatory processes, many pathological conditions and underlying processes of chronic age-related diseases. Therefore there is increased expectation that therapeutics can be developed which act in some fashion to suppress reactive oxidant species and ameliorate the condition. This has turned out to be more difficult than at first expected. Developing therapeutics for indications where reactive oxidant species is an important consideration presents some unique challenges. We discuss important questions including whether reactive oxidant species should be a therapeutic target and the need to recognize the fact that an antioxidant in a defined chemical system may be a poor antioxidant operationally in a biological system and the importance of considering the fact that reactive oxidant species may accompany the disease or pathological system rather than being a causative factor. We also discuss the value of having preclinical models to determine if the processes which are important in causing the disease under study is critically dependent on reactive oxidant species events and if the therapeutic under consideration quells these processes. In addition we discuss measures of success that must be met in commercial research and development in preclinical and clinical trials and discuss as examples our translational research effort in developing nitrones for the treatment of acute ischemic stroke and as anti-cancer agents.

Ventricular cerebrospinal fluid (vCSF) obtained at autopsy from 230 participants in the Religious Orders Study was analyzed for alpha tocopherol (αT, vitamin E) and gamma tocopherol (γT) in relation to brain tissue neuropathological diagnoses (NIA-Reagan criteria); neuritic plaque density and neurofibrillary tangle state (Braak stage); and cognitive function proximate to death. Neither vCSF αT nor γT was related to the pathological diagnosis of Alzheimer’s disease, but vCSF αT concentration was inversely related to neuritic plaque density (β = −0.21, SE = 0.105, p = 0.04) in regression models adjusted for age, gender, education, and APOE-4. Ventricular CSF αT concentration was positively associated with perceptual speed (β = 0.27, SE = 0.116, p = 0.02) whereas the γT/αT ratio was negatively associated with episodic memory (β = −0.037, SE = 0.017, p = 0.04). Only vCSF αT, but not γT, was correlated with postmortem interval (PMI). Adjustment for PMI had no effect on significance of associations between αT and perceptual speed or γT/αT and episodic memory, but after this adjustment the αT concentration was no longer significantly associated with neuritic plaques. These data suggest that vCSF αT, but not γT, is weakly associated with less Alzheimer’s disease neuropathology, specifically neuritic plaques, and correlates with better performance on tests of perceptual speed.