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1.
ASN Neuro ; 14: 17590914221112352, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35791633

RESUMO

Although over 20 disease modifying therapies are approved to treat Multiple Sclerosis (MS), these do not increase remyelination of demyelinated axons or mitigate axon damage. Previous studies showed that lanthionine ketenamine ethyl ester (LKE) reduces clinical signs in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and increased maturation of oligodendrocyte (OL) progenitor cells (OPCs) in vitro. In the current study, we used the cuprizone (CPZ) demyelination model of MS to test if LKE could increase remyelination. The corpus callosum (CC) and somatosensory cortex was examined by immunohistochemistry (IHC), electron microscopy and for mRNA expression changes in mice provided 5 weeks of CPZ diet followed by 2 weeks of normal diet in the presence of LKE or vehicle. A significant increase in the number of myelinated axons, and increased myelin thickness was observed in the CC of LKE-treated groups compared to vehicle-treated groups. LKE also increased myelin basic protein and proteolipid protein expression in the CC and cortex, and increased the number of mature OLs in the cortex. In contrast, LKE did not increase the percentage of proliferating OPCs suggesting effects on OPC survival and differentiation but not proliferation. The effects of LKE on OL maturation and remyelination were supported by similar changes in their relative mRNA levels. Interestingly, LKE did not have significant effects on GFAP or Iba1 immunostaining or mRNA levels. These findings suggest that remyelinating actions of LKE can potentially be formulated to induce remyelination in neurological diseases associated with demyelination including MS.


Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Remielinização , Aminoácidos Sulfúricos , Animais , Cuprizona/toxicidade , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Ésteres/farmacologia , Camundongos , Esclerose Múltipla/tratamento farmacológico , RNA Mensageiro
2.
Neurochem Res ; 47(8): 2373-2382, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35589915

RESUMO

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 GSK3ß in the hippocampus is related to pathology and memory impairment in DLB, and LKE may have clinical implications in the treatment of α-synucleinopathy.


Assuntos
Aminoácidos Sulfúricos , Sinucleinopatias , Aminoácidos Sulfúricos/farmacologia , Aminoácidos Sulfúricos/uso terapêutico , Animais , Modelos Animais de Doenças , Ésteres , Humanos , Camundongos , Camundongos Transgênicos , alfa-Sinucleína
3.
Free Radic Biol Med ; 178: 161-173, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34863876

RESUMO

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.


Assuntos
Doenças Neuroinflamatórias , Proteostase , Glutationa/metabolismo , Humanos , Oxirredução , Estresse Oxidativo , Transdução de Sinais , Enxofre
4.
Free Radic Biol Med ; 161: 115-124, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33049334

RESUMO

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.


Assuntos
Enzimas Desubiquitinantes , Peróxido de Hidrogênio , Receptores Acoplados a Proteínas G , Células HeLa , Humanos , Hidroliases , Complexos Multienzimáticos , Oxirredução , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina Tiolesterase
5.
J Neurol Sci ; 413: 116802, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32244093

RESUMO

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.


Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Doença de Parkinson , Aminoácidos Sulfúricos , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos , Ésteres , Camundongos , Camundongos Endogâmicos C57BL , Microglia , Fosforilação , Substância Negra
8.
Genes Cells ; 24(1): 31-40, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30375127

RESUMO

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by slow and progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (l-Dopa), the current main treatment for PD, supplies dopamine, but it does not prevent neurodegeneration. There is thus no promising remedy for PD. Recent in vitro study showed the increase in the phosphorylation levels of Collapsin Response Mediator Protein 2 (CRMP2) is involved in dopaminergic axon degeneration. In the present study, we report elevation of CRMP2 phosphorylation in dopaminergic neurons in SNc after challenge with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a common model for PD. Genetic suppression of CRMP2 phosphorylation by mutation of the obligatory Cyclin-dependent kinase 5 (Cdk5)-targeted serine-522 site prevented axonal degradation in the nigrostriatal pathway of transgenic mice. As a result, the degree of MPTP-induced motor impairment in the rotarod test was suppressed. These results suggest that suppression of CRMP2 phosphorylation may be a novel therapeutic target for PD.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Supressão Genética , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Atividade Motora , Neostriado/patologia , Degeneração Neural/patologia , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Fosforilação , Substância Negra/patologia
9.
J Biol Chem ; 293(47): 18242-18269, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30282635

RESUMO

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 ß-amyloid (Aß) 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.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/efeitos dos fármacos , Polissacarídeos Bacterianos/administração & dosagem , Administração Intranasal , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/genética , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
10.
J Chromatogr A ; 1573: 66-77, 2018 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-30201162

RESUMO

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.


Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida de Alta Pressão , Microcistinas/sangue , Microcistinas/urina , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Urinálise/métodos , Animais , Humanos , Camundongos
11.
Rapid Commun Mass Spectrom ; 32(22): 1941-1948, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30117207

RESUMO

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.


Assuntos
Aminoácidos Sulfúricos/sangue , Aminoácidos Sulfúricos/farmacocinética , Encéfalo/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão/métodos , Ésteres/sangue , Ésteres/farmacocinética , Limite de Detecção , Camundongos
12.
Bioorg Med Chem Lett ; 28(4): 562-565, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29398540

RESUMO

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.


Assuntos
Aminoácidos Sulfúricos/farmacologia , Cicloexenos/farmacologia , Ésteres/farmacologia , Ácidos Fosforosos/farmacologia , Tiazinas/farmacologia , Aminoácidos Sulfúricos/síntese química , Animais , Autofagia/efeitos dos fármacos , Células CACO-2 , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cicloexenos/síntese química , Ésteres/síntese química , Humanos , Macrolídeos/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Ácidos Fosforosos/síntese química , Ratos , Tiazinas/síntese química
13.
Neurochem Int ; 115: 61-68, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29475037

RESUMO

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.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Aminoácidos Sulfúricos/farmacologia , Neuroproteção/efeitos dos fármacos , Ácido Okadáico/farmacologia , Doença de Alzheimer/induzido quimicamente , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Feminino , Masculino , Camundongos , Fármacos Neuroprotetores/farmacologia , Peixe-Zebra
14.
Mol Cell Biochem ; 442(1-2): 29-38, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28913673

RESUMO

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.


Assuntos
Proteínas ADAM/metabolismo , Integrina alfa4/metabolismo , Proteínas ADAM/genética , Animais , Células CHO , Adesão Celular/fisiologia , Cricetulus , Humanos , Integrina alfa4/genética , Células Jurkat , Células K562 , Ligantes
15.
Neurosci Lett ; 664: 28-33, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29128626

RESUMO

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.


Assuntos
Aminoácidos Sulfúricos/farmacologia , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Neurônios/fisiologia , Neuroproteção/fisiologia
16.
Amino Acids ; 49(4): 747-759, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28044206

RESUMO

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.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/química , Proteínas do Tecido Nervoso/química , Processamento de Proteína Pós-Traducional , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química
17.
Biomarkers ; 22(3-4): 372-382, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28055279

RESUMO

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) (<10 kDa) SDS-PAGE bands exhibited changes after stress. DISCUSSION AND CONCLUSION: Alpha-amylase and cystatins are potential salivary markers of acute stress, but further validation should be performed using larger sample populations.


Assuntos
Proteômica/métodos , Proteínas e Peptídeos Salivares/metabolismo , Estresse Psicológico/metabolismo , Adulto , Eletroforese em Gel de Poliacrilamida , Serviços Médicos de Emergência/métodos , Feminino , Humanos , Internato e Residência , Masculino , Espectrometria de Massas , Projetos Piloto , Cistatinas Salivares/análise , Proteínas e Peptídeos Salivares/análise , Adulto Jovem , alfa-Amilases/análise
18.
Biochem Biophys Res Commun ; 483(1): 759-764, 2017 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-27965088

RESUMO

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.


Assuntos
Aminoácidos Sulfúricos/uso terapêutico , Anti-Inflamatórios não Esteroides/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Medula Espinal/fisiopatologia , Aminoácidos Sulfúricos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Axônios/efeitos dos fármacos , Axônios/fisiologia , Biomarcadores/análise , Células Cultivadas , Modelos Animais de Doenças , Feminino , Inflamação/tratamento farmacológico , Inflamação/patologia , Locomoção/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/fisiologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Serotonina/metabolismo , Medula Espinal/efeitos dos fármacos , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia
19.
J Alzheimers Dis ; 53(1): 1-14, 2016 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-27079722

RESUMO

Alzheimer's disease (AD) has long been viewed as a pathology that must be caused either by aberrant amyloid-ß protein precursor (AßPP) processing, dysfunctional tau protein processing, or a combination of these two factors. This is a reasonable assumption because amyloid-ß peptide (Aß) accumulation and tau hyperphosphorylation are the defining histological features in AD, and because AßPP 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.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas tau/metabolismo , Animais , Humanos
20.
PLoS One ; 11(3): e0149715, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26939023

RESUMO

An enormous amount of efforts have been poured to find an effective therapeutic agent for the treatment of neurodegenerative diseases including Alzheimer's disease (AD). Among those, neurotrophic peptides that regenerate neuronal structures and increase neuron survival show a promise in slowing neurodegeneration. However, the short plasma half-life and poor blood-brain-barrier (BBB)-permeability of neurotrophic peptides limit their in vivo efficacy. Thus, an alternative neurotrophic agent that has longer plasma half-life and better BBB-permeability has been sought for. Based on the recent findings of neuroprotective polysaccharides, we searched for a BBB-permeable neuroprotective polysaccharide among natural polysaccharides that are approved for human use. Then, we discovered midi-GAGR, a BBB-permeable, long plasma half-life, strong neuroprotective and neurotrophic polysaccharide. Midi-GAGR is a 4.7kD cleavage product of low acyl gellan gum that is approved by FDA for human use. Midi-GAGR protected rodent cortical neurons not only from the pathological concentrations of co-/post-treated free reactive radicals and Aß42 peptide but also from activated microglial cells. Moreover, midi-GAGR showed a good neurotrophic effect; it enhanced neurite outgrowth and increased phosphorylated cAMP-responsive element binding protein (pCREB) in the nuclei of primary cortical neurons. Furthermore, intra-nasally administered midi-GAGR penetrated the BBB and exerted its neurotrophic effect inside the brain for 24 h after one-time administration. Midi-GAGR appears to activate fibroblast growth factor receptor 1 (FGFR1) and its downstream neurotrophic signaling pathway for neuroprotection and CREB activation. Additionally, 14-day intranasal administration of midi-GAGR not only increased neuronal activity markers but also decreased hyperphosphorylated tau, a precursor of neurofibrillary tangle, in the brains of the AD mouse model, 3xTg-AD. Taken together, midi-GAGR with good BBB-permeability, long plasma half-life, and strong neuroprotective and neurotrophic effects has a great therapeutic potential for the treatment of neurodegenerative diseases, especially AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Polissacarídeos Bacterianos/administração & dosagem , Polissacarídeos/uso terapêutico , Doença de Alzheimer/patologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Sobrevivência Celular , Humanos , Camundongos , Degeneração Neural/tratamento farmacológico , Degeneração Neural/patologia , Fármacos Neuroprotetores/farmacocinética , Polissacarídeos/farmacocinética
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