Search

Potentially druggable mechanisms underlying synaptic deficits seen in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are under intense interrogations. In addition to defective synaptic vesicle trafficking, cytoskeletal disruption, autophagic perturbation, and neuroinflammation, hyperphosphorylation of microtubule-associated protein collapsin response mediator protein 2 (CRMP2, also known as DPYSL2) is newly determined to correlate with synaptic deficits in human DLB. The small molecule experimental therapeutic, lanthionine ketimine-5-ethyl ester (LKE), appears to interact with CRMP2 in a host of neurodegenerative mouse models, normalizing its phosphorylation level while promoting healthful autophagy in cell culture models and suppressing the proinflammatory phenotype of activated microglia. Accordingly, this study examined the effect of LKE on α-synuclein A53T transgenic (Tg) mice which were employed as a DLB model. We found that chronic administration of LKE to A53T mice suppressed (1) the accumulation of LBs, (2) neuroinflammatory activation of microglia, (3) impairment of contextual fear memory, and (4) CRMP2 phosphorylation at Thr509 in A53T Tg mice. These results suggest that CRMP2 phosphorylation by GSK3β in the hippocampus is related to pathology and memory impairment in DLB, and LKE may have clinical implications in the treatment of α-synucleinopathy.

Although we can thoroughly describe individual neurodegenerative diseases from the molecular level through cell biology to histology and clinical presentation, our understanding of them and hence treatment gains have been depressingly limited, partly due to difficulty conceptualizing different diseases as variations within the same overarching pathological rubric. This review endeavors to create such rubric by knitting together the seemingly disparate phenomena of oxidative stress, dysregulated proteostasis, and neuroinflammation into a cohesive triad that highlights mechanistic connectivities. We begin by considering that brain metabolic demands necessitate careful control of oxidative homeostasis, largely through sulfur redox chemistry and glutathione (GSH). GSH is essential for brain antioxidant defense, but also for redox signaling and thus neuroinflammation. Delicate regulation of neuroinflammatory pathways (NFκB, MAPK-p38, and NLRP3 particularly) occurs through S-glutathionylation of protein phosphatases but also through redox-sensing elements like ASK1; the 26S proteasome and cysteine deubiquitinases (DUBs). The relationship amongst triad elements is underscored by our discovery that LanCL1 (lanthionine synthetase-like protein-1) protects against oxidant toxicity; mediates GSH-dependent reactivation of oxidized DUBs; and antagonizes the pro-inflammatory cytokine, tumor necrosis factor-α (TNFα). We highlight currently promising pharmacological efforts to modulate key triad elements and suggest nexus points that might be exploited to further clinical advantage.

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.

RATIONALE Preclinical studies in the search for treatments for several neurodegenerative diseases have identified lanthionine ketimine (LK) and its monoethyl ester derivative (LKE) as potential candidates. An ultrahigh-pressure liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) assay was developed to evaluate bioavailability by measuring these compounds in mouse serum, whole blood and brain tissue. METHODS Following administration of LKE to mice for 3 days in chow at 300 ppm, the animals were sacrificed, and LKE was extracted from serum, whole blood and brain tissues through protein precipitation using cold methanol. To enhance chromatographic separation and electrospray ionization, LK was methylated using diazomethane. Separations were carried out using C18 reversed-phase UHPLC, and quantitative measurements were obtained using on-line triple-quadruple mass spectrometry with positive ion electrospray ionization, collision-induced dissociation and selected reaction monitoring. Tolbutamide was used as internal standard. RESULTS LKE showed good recovery ranging from 77-90% in serum and 82-88% in brain tissue. An eight-point standard curve ranging from 0.005 to 4.6 μM was linear (R2 0.998). The average LKE detected in mouse serum was 277.42 nM, while the concentration in whole blood was 38 nM. Neither LK nor LKE was detected in brain tissues. CONCLUSIONS A rapid quantitative method to measure LKE in mouse serum, whole blood and brain tissues using UHPLC/MS/MS was developed and validated following FDA guidelines. This method is suitable for bioavailability and pharmacokinetic studies.

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.

Lanthionine ketimine ethyl ester (LKE) is a synthetic derivative of the naturally occurring amino acid lanthionine ketimine. We previously showed that LKE reduced clinical signs in a mouse model of multiple sclerosis (MS) associated with reductions in axonal damage; however, whether LKE has direct beneficial actions on mammalian neuronal cells was not examined. In the current study, we tested the effects of LKE in SH-SY5Y human neuronal cells and in primary mouse cerebellar granule neurons. In both cell types, LKE dose-dependently reduced the cell death that occurred spontaneously followed a change in media. LKE also reduced cell death due to glutamate excitoxicity, accompanied by a reduction in production of reactive oxygen species. LKE induced neuritogenesis in both undifferentiated SH-SY5Y cells and in primary neuron, increasing process numbers and lengths. These results demonstrate that direct neuroprotective and neurotrophic effects of LKE likely contribute to its beneficial actions in vivo.

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.

The mammalian central nervous system (CNS) has limited regenerative ability after injury, largely due to scar formation and axonal growth inhibitors. Experimental suppression of neuroinflammation encourages recovery from spinal cord injury (SCI), yet practical means for pharmacologically treating SCI have remained elusive. Lanthionine ketimine (LK) is a natural brain sulfur amino acid metabolite with demonstrated anti-neuroinflammatory and neurotrophic activities. LK and its synthetic brain-penetrating ethyl ester (LKE) promote growth factor-dependent neurite extension in cultured cell and suppress microglial activation in animal models of neurodegeneration. Thus far however, LKE has not been explored as a potential therapy for SCI. The present study investigated the hypothesis that systemic LKE could improve motor functional recovery after SCI in a mouse model. Intraperitoneal administration of LKE (100 mg/kg/d) after near-complete transect of spinal cord at the T7 level significantly improved motor function over a 4-week time course. Vehicle-treated mice, in contrast, demonstrated negligible functional recovery. In terms of histology, LKE treatment reduced pro-neuroinflammatory microglia/macrophage activation evidenced by quantitative Iba1 labeling and shifted the microglial phenotype toward a more neurotrophic M2 character evidenced by changes in the M2 marker arginase-1. This was correlated with less dense scar formation and more extensive axonal regrowth across the transection site demonstrated by 5-hydroxytryptamine (5HT) immunolabeling of raphespinal tract axons. These data provide evidence that LKE or similar compounds have potential therapeutic value for recovery after certain forms of SCI.

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.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (L-Dopa), the current main treatment for PD, reduces PD symptoms by partially replacing dopamine, but it does not slow neurodegeneration. Recent studies have evidenced that neuroinflammatory processes contribute to the degeneration of dopaminergic neurons in the SNc under cytopathic conditions, while other lines of inquiry have implicated phosphorylation of collapsin response mediator protein 2 (CRMP2) as a causal factor in axonal retraction after neural injury. We recently reported on the therapeutic effect of lanthionine ketimine ester (LKE) which associates with CRMP2 following axonal injury in the spinal cord. In the present study, we report that LKE protects SNc dopaminergic neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) challenge, a common model for PD, and reduces the number of activated microglia proximal to the damaged SNc. The results also show that MPTP-induced motor impairment was suppressed in LKE treatment. Furthermore, the results show that LKE inhibits the elevation of CRMP2 phosphorylation in dopaminergic neurons in the SNc after MPTP injection. These data suggest that modification of CRMP2 phosphorylation and suppression of microglial activation with LKE administration may represent a novel strategy for slowing progress of pathological processes in PD.

[Image: see text]

The protocols for solid-phase extraction (SPE) of six microcystins (MCs; MC-LR, MC-RR, MC-LA, MC-LF, MC-LW, and MC-YR) from mouse urine, mouse plasma, and human serum are reported. The quantification of those MCs in biofluids was achieved using HPLC-orbitrap-MS in selected-ion monitoring (SIM) mode, and MCs in urine samples were also quantified by ultra-HPLC-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS) in multiple reaction monitoring (MRM) mode. Under optimal conditions, the extraction recoveries of MCs from samples spiked at two different concentrations (1 μg/L and 10 μg/L) ranged from 90.4% to 104.3% with relative standard deviations (RSDs) ≤ 4.7% for mouse urine, 90.4-106.9% with RSDs ≤ 6.3% for mouse plasma, and 90.0-104.8% with RSDs ≤ 5.0% for human serum. Matrix-matched internal standard calibration curves were linear with R2 ≥ 0.9950 for MC-LR, MC-RR and MC-YR, and R2 ≥ 0.9883 for MC-LA, MC-LF, and MC-LW. The limits of quantification (LOQs) in spiked urine samples were ∼0.13 μg/L for MC-LR, MC-RR, and MC-YR, and ∼0.50 μg/L for MC-LA, MC-LF, and MC-LW, while the LOQs in spiked plasma and serum were ∼0.25 μg/L for MC-LR, MC-RR, and MC-YR, and ∼1.00 μg/L for MC-LA, MC-LF, and MC-LW. The developed methods were applied in a proof-of-concept study to quantify urinary and blood concentrations of MC-LR after oral administration to mice. The urine of mice administered 50 μg of MC-LR per kg bodyweight contained on average 1.30 μg/L of MC-LR (n = 8), while mice administered 100 μg of MC-LR per kg bodyweight had average MC-LR concentration of 2.82 μg/L (n = 8). MC-LR was also quantified in the plasma of the same mice. The results showed that increased MC-LR dosage led to larger urinary and plasma MC-LR concentrations and the developed methods were effective for the quantification of MCs in mouse biofluids.

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.

Free radicals associated with oxidative stress play a major role in amyotrophic lateral sclerosis (ALS). By combining immuno-spin trapping and molecular magnetic resonance imaging, in vivo trapped radical adducts were detected in the spinal cords of SOD1(G93A)-transgenic (Tg) mice, a model for ALS. For this study, the nitrone spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide) was administered (ip) over 5 days before administration (iv) of an anti-DMPO probe (anti-DMPO antibody covalently bound to an albumin-gadolinium-diethylenetriamine pentaacetic acid-biotin MRI contrast agent) to trap free radicals. MRI was used to detect the presence of the anti-DMPO radical adducts by a significant sustained increase in MR signal intensities (p < 0.05) or anti-DMPO probe concentrations measured from T₁ relaxations (p < 0.01). The biotin moiety of the anti-DMPO probe was targeted with fluorescence-labeled streptavidin to locate the probe in excised tissues. Negative controls included either Tg ALS mice initially administered saline rather than DMPO followed by the anti-DMPO probe or non-Tg mice initially administered DMPO and then the anti-DMPO probe. The anti-DMPO probe was found to bind to neurons via colocalization fluorescence microscopy. DMPO adducts were also confirmed in diseased/nondiseased tissues from animals administered DMPO. Apparent diffusion coefficients from diffusion-weighted images of spinal cords from Tg mice were significantly elevated (p < 0.001) compared to wild-type controls. This is the first report regarding the detection of in vivo trapped radical adducts in an ALS model. This novel, noninvasive, in vivo diagnostic method can be applied to investigate the involvement of free radical mechanisms in ALS rodent models.

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.

Lanthionine ketimine (LK) is a natural sulfur amino acid metabolite with potent neurotrophic activity. Proteomics indicate that LK interacts with collapsin response mediator protein-2 (CRMP2/DPYSL2/UNC-33), a brain-enriched protein that was shown to regulate cytoskeletal remodeling, neuronal morphology, and synaptic function. To elucidate further the molecular interplay and biological action of LK and UNC-33, we began examining the nervous system of Caenorhabditis elegans nematodes in which both LK concentrations and UNC-33 protein were manipulated. To this end, a cell-permeable LK-ester (LKE) was administered to developing C. elegans engineered to express yellow fluorescent protein (YFP) in cholinergic neurons (strain RM3128) or green fluorescent protein (GFP) in GABAergic neurons (strain CZ1200), and neural morphology was assessed. Fluorescent imaging analyses show that LKE exposure to wild-type animals induced neural commissure outgrowth, crossing over, and bundling in both neurites from GABAergic and cholinergic motor neurons. Additionally, when unc-33(e204) hypomorph mutant nematodes (D389N substitution mutants) were exposed to LKE, both the neuroanatomical defects of incomplete dorsoventral neural commissures and the ventral nerve cord gaps were partially rescued. In contrast, LKE did not rescue ventral nerve cord gaps found in unc-33(mn407) null mutant. Together these data suggest possible functions for LK as a regulator of neuritic elongation, corroborate roles for UNC-33/CRMP2 in the mechanism of LKE activity, and suggest the potential of LKE as a therapeutic molecule for neurological diseases involving CRMP2 dysfunction.

The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM-integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these "dead" enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.

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.

Collapsin response mediator protein-2 (DPYSL2 or CRMP2) is a multifunctional adaptor protein within the central nervous system. In the developing brain or cell cultures, CRMP2 performs structural and regulatory functions related to cytoskeletal dynamics, vesicle trafficking and synaptic physiology whereas CRMP2 functions in adult brain are still being elucidated. CRMP2 has been associated with several neuropathologic or psychiatric conditions including Alzheimer's disease (AD) and schizophrenia, either at the level of genetic polymorphisms; protein expression; post-translational modifications; or protein/protein interactions. In AD, CRMP2 is phosphorylated by glycogen synthase kinase-3β (GSK3β) and cyclin dependent protein kinase-5 (CDK5), the same kinases that act on tau protein in generating neurofibrillary tangles (NFTs). Phosphorylated CRMP2 collects in NFTs in association with the synaptic structure-regulating SRA1/WAVE1 (specifically Rac1-associated protein-1/WASP family verprolin-homologous protein-1) complex. This phenomenon could plausibly contribute to deficits in neural and synaptic structure that have been well documented in AD. This review discusses the essential biology of CRMP2 in the context of nascent data implicating CRMP2 perturbations as either a correlate of, or plausible contributor to, diverse neuropathologies. A discussion is made of recent findings that the atypical antidepressant tianeptine increases CRMP2 expression, whereas other, neuroactive small molecules including the epilepsy drug lacosamide and the natural brain metabolite lanthionine ketimine appear to bind CRMP2 directly with concomitant affects on neural structure. These findings constitute proofs-of-concept that pharmacological manipulation of CRMP2 is possible and hence, may offer new opportunities for therapy development against certain neurological diseases.

Background Peripheral arterial disease (PAD) causes narrowing of blood vessels resulting in restriction of blood flow to lower limbs. Recently, autophagy has progressed as an emerging therapeutic strategy targeted to minimize the risk of PAD. Autophagy is a catabolic process aimed at recycling damaged proteins and organelles. Cystathionine-γ-lyase (CSE) is a major H2S producing enzyme in the vasculature, which is vasoprotective. However, the role of CSE in regulating autophagy remains unknown. Hypothesis CSE derived sulfide metabolites protect endothelial cells from ischemic injury by inducing autophagy. Methods WT and CSE knockout mice were subjected to femoral artery ligation (FAL). Mouse aortic endothelial cells (MAECs) isolated from WT and CSE-/- mice served as in vitro model. Autophagic flux was measured with and without chloroquine (50mg/kg) and bafilomycin (50nM) in animals and cells respectively. Trypan blue assay was used to evaluate cell viability. Annexin V/ PI staining and the cleaved caspase-3 assay were used to evaluate apoptosis. Results In WT and CSE-/- mice subjected to FAL, CSE-/- mice showed defective autophagy. MAECs deficient in CSE-/- also showed impaired autophagy. Transfection with mRFP-GFP tandem fluorescent-tagged LC3 plasmid showed increased autolysosomes in WT compared to CSE-/- MAEC’s. Interestingly, re-expression of human CSE in CSE-/- endothelial cells restored autophagy. Exogenous sulfide treatment, including per/polysulfides were not able to restore autophagy in CSE-/- cells. However, lanthionine ketimine (LK), a novel sulfur metabolite of the transulfuration pathway, restored autophagy in its cell permeable ester form. Lastly, we observed that deficiency of CSE blocks autophagy leading to apoptosis in endothelial cells. Conclusion We reveal for the first time that LK, a CSE derived sulfur metabolite, could regulate autophagy in ischemic endothelial cells. These findings reveal a potential new experimental approach to protect against ischemic injury by activating autophagy during PAD.

Lanthionine cyclase-like protein-1 (LanCL1) is a glutathione-binding Zn2+ metalloprotein present ubiquitously in mammalian cells and expressed heavily in brain and kidney. LanCl1 function has not been exactly elucidated. Recent reports contend that LanCL1 genetic ablation induced age-related neurodegeneration in mice. Some evidence has been published that LanCL1 has glutathione-S-transferase (GST)-like antioxidant activity, while other recent claims have been made that LanCL1 suppresses phospho-activation of c-Jun amino-terminal kinase (JNK) and other mitogen-activated kinase (MAPK) cascades; but none of these prior studies has been independently replicated, to date. In this study, CRISPR-Cas9 technology was used to completely and stably ablate LanCL1 from HeLa cells and the cells were then phenotyped with respect to their behavior in the face of an oxidative challenge. LanCL1-KO cells appeared grossly normal but were hypersensitive to H2O2 toxicity (LD50% = 171 μM for wild-type vs 87 μM for LanCL1-KO cells). Cellular [GSH], [GSSG], and [GSH]/[GSH] were unaltered by LanCL1 knockout; however, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were significantly elevated and GST activity was non-significantly elevated in LanCL1 KO cultures. LanCL1 KO cells showed no alterations in basal or H2O2 –induced phosphorylation of p38-MAPK, ERK p42/p44 or JNK. There was, however, a significant 52% decrease in total c-Jun and a 28% decrease in NFKB proteins associated with LanCL1-KO. Taken together these data suggest that LanCL1 likely is not a GST-like antioxidant enzyme itself, but works to regulate redox-sensitive transcription factor activation downstream from classic MAPK cascades. The data emphasize that LanCL1 expression is vital to maintenance of cell homeostasis in the face of a potentially toxic redox challenge, and underscore the importance of elucidating the exact function of this novel glutathione-binding protein. Research supported in part by National Institutes of Health (NS093594; NS082283) and the Degen Foundation.

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 objective of this study was to determine the composition and content of sesamin and desmethyl tocopherols such as alpha-tocopherol (alphaT), delta-tocopherol (deltaT) and gamma-tocopherol (gammaT) in seeds of sesame (Sesamum indicum L.) for 11 genotypes conserved in the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia, USA. Seed accessions studied were collections from eight countries worldwide, including one landrace from Thailand and two cultivars from Texas, USA. Novel methodologies and analytical techniques described herein consisted of reverse-phase high-performance liquid chromatography (HPLC) connected in series with two detection systems specific for each analyte class. Photodiode array detection was employed for sesamin analysis and electrochemical array detection was used in the determination of tocopherols. A preliminary study was conducted to assess sesamin levels in 2003 and tocopherol levels in 2004 from sesame seed samples conserved at the USDA, ARS and PGRCU. In 2005, sesame seed samples were grown, harvested and evaluated for sesamin as well as tocopherol levels. The overall results (n = 3) showed that sesamin, alphaT, deltaT and gammaT levels were 0.67-6.35 mg/g, 0.034-0.175 microg/g, 0.44-3.05 microg/g and 56.9-99.3 microg/g respectively, indicating that the sesame seed accessions contained higher levels of sesamin and gammaT compared with alphaT and deltaT. Statistical analysis was conducted and significant differences were observed among the 11 different sesame genotypes. This suggests that genetic, environmental and geographical factors influence sesamin and desmethyl tocopherol content.

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.