GlyT1 Inhibition by NFPS Promotes Neuroprotection in Amyloid-ß-Induced Alzheimer's Disease Animal Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-ß, leading to N-methyl-D-aspartate (NMDA) receptor-dependent synaptic depression, spine elimination, and memory deficits. Glycine transporter type 1 (GlyT1) modulates glutamatergic neurotransmission via NMDA receptors (NMDAR), presenting a potential alternative therapeutic approach for AD. This study investigates the neuroprotective potential of GlyT1 inhibition in an amyloid-ß-induced AD mouse model. C57BL/6 mice were treated with N-[3-([1,1-Biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine (NFPS), a GlyT1 inhibitor, 24 h prior to intrahippocampal injection of amyloid-ß. NFPS pretreatment prevented amyloid-ß-induced cognitive deficits in short-term and long-term memory, evidenced by novel object recognition and spatial memory tasks. Moreover, NFPS pretreatment curbed microglial activation, astrocytic reactivity, and subsequent neuronal damage from amyloid-ß injection. An extensive label-free quantitative UPLC-MSE proteomic analysis was performed on the hippocampi of mice treated with NFPS. In proteomics, KEGG enrichment analysis revealed increased in dopaminergic synapse, purine-containing compound biosynthetic process and long-term potentiation, and a reduction in Glucose catabolic process and glycolytic process pathways. The western blot analysis confirmed that NFPS treatment elevated BDNF levels, correlating with enhanced TRKB phosphorylation and mTOR activation. Moreover, NFPS treatment reduced the GluN2B expression after 6 h, which was associated with an increase on CaMKIV and CREB phosphorylation. Collectively, these findings demonstrate that GlyT1 inhibition by NFPS activates diverse neuroprotective pathways, enhancing long-term potentiation signaling and countering amyloid-ß-induced hippocampal damage.

Sarcosine (glycine transporter inhibitor) attenuates behavioural and biochemical changes induced by ketamine, in the rat model of schizophrenia.

Schizophrenia is a neurological disorder that alters the behavior and affects the quality of life of a patient. It is characterized by hallucinations, disorganized behavior, cognitive dysfunction, hyperlocomotion, and loss of the reward system. Schizophrenia constitutes three symptoms' domains, viz. positive, negative and cognitive. Typical and atypical antipsychotics do not fully resolve all the symptoms' domains thus paving the way to the genesis of the glutamatergic hypothesis, i.e. N-methyl-D-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Positive modulation of NMDA receptors by enhancing co-agonist, glycine effect is proposed to produce a therapeutic effect in schizophrenia. Hence, sarcosine (N-methyl glycine), natural amino acid, and a glycine transporter inhibitor (GlyT-1) which also acts on NMDA receptors were used in the present study. The present study unravels the role of sarcosine in the attenuation of ketamine-induced three symptom domains in a rat model through modulation of oxidative stress, mitochondrial dysfunction, and neuroinflammatory pathways. The animal model of schizophrenia was established by injecting ketamine intraperitoneal (ip) at a 30 mg/kg dose for 10 consecutive days, after which sarcosine (300, 600 mg/kg, ip) as a treatment was given for 7 days followed by behavioral, biochemical, molecular, and histopathological analysis. It was revealed that sarcosine reversed ketamine-induced behavioral impairments. Moreover, sarcosine ameliorated oxidative and nitrosative stress, mitochondrial dysfunction, and neuroinflammation and showed protective effects in histopathological examination by hematoxylin and eosin staining. Hence, conclusively, sarcosine was regarded to attenuate the behavioural symptoms of schizophrenia by alleviating oxidative stress, neuroinflammation, and mitochondrial dysfunction established by the ketamine.

Sarcosine sensitivity in Escherichia coli is mediated by activation of the glycine cleavage system.

Corynebacterium glutamicum AJ1511 and Escherichia coli BW25113 strains were compared in terms of resistance to sarcosine (N-methylglycine). The E. coli strain was more sensitive to sarcosine than C. glutamicum, especially when grown in minimal medium. Growth inhibition of the BW25113 strain in minimal M9 medium containing 0.5 m sarcosine was overcome by the addition of glycine. Inactivation of the glycine cleavage (GCV) system (∆gcvP) as well as the removal of its activator (∆gcvA) in BW25113 cells increased the threshold for sarcosine inhibition up to 0.75 m. Activation of the promoter of the E. coli gcvTHP operon by 0.1-0.4 m sarcosine added to M9 medium was demonstrated in vivo using dasherGFP as the reporter. Sensitivity to sarcosine on glucose minimal medium is suggested to be a characteristic of Gram-negative bacteria with GcvA/GcvR regulation of the GCV system.

ERVK13-1/miR-873-5p/GNMT Axis Promotes Metastatic Potential in Human Bladder Cancer though Sarcosine Production.

N-methyl-glycine (sarcosine) is known to promote metastatic potential in some cancers; however, its effects on bladder cancer are unclear. T24 cells derived from invasive cancer highly expressed GNMT, and S-adenosyl methionine (SAM) treatment increased sarcosine production, promoting proliferation, invasion, anti-apoptotic survival, sphere formation, and drug resistance. In contrast, RT4 cells derived from non-invasive cancers expressed low GNMT, and SAM treatment did not produce sarcosine and did not promote malignant phenotypes. In T24 cells, the expression of miR-873-5p, which suppresses GNMT expression, was suppressed, and the expression of ERVK13-1, which sponges miR-873-5p, was increased. The growth of subcutaneous tumors, lung metastasis, and intratumoral GNMT expression in SAM-treated nude mice was suppressed in T24 cells with ERVK13-1 knockdown but promoted in RT4 cells treated with miR-873-5p inhibitor. An increase in mouse urinary sarcosine levels was observed to correlate with tumor weight. Immunostaining of 86 human bladder cancer cases showed that GNMT expression was higher in cases with muscle invasion and metastasis. Additionally, urinary sarcosine concentrations increased in cases of muscle invasion. Notably, urinary sarcosine concentration may serve as a marker for muscle invasion in bladder cancer; however, further investigation is necessitated.

Structure-activity relationship study of amphipathic antimicrobial peptides using helix-destabilizing sarcosine.

Antimicrobial peptides (AMPs) are potential therapeutic agents against bacteria. We recently showed that a rationally designed AMP, termed Stripe, with an amphipathic distribution of native cationic and hydrophobic amino acids on its helical structure exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria with negligible hemolytic activity and cytotoxicity. In this study, the structure-activity relationship of Stripe was elucidated by designing a series of antimicrobial peptides whereby amino acid residues of Stripe were exchanged with helix-destabilizing sarcosine residues. Stripe 1-5 peptides with hydrophobic amino acids substituted with sarcosine were predominantly unstructured and showed no antimicrobial activity, except against Escherichia coli (E. coli) (DH5α) cells. The activity against E. coli (DH5α) cells and the helicity of Stripe 1-5 peptides decreased concomitantly as the number of sarcosine residue substitutions increased. Stripe 1-5 peptides showed no hemolytic activity or cytotoxicity. The results indicate that sarcosine substitutions provide an approach to study the structure-activity relationship of helical AMPs, and the helicity of Stripe is an important feature defining its activity.

Pharmacological Evidence on Augmented Antiallodynia Following Systemic Co-Treatment with GlyT-1 and GlyT-2 Inhibitors in Rat Neuropathic Pain Model.

The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30-60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP.

Ex vivo anti-microbial efficacy of various formaldehyde releasers against antibiotic resistant and antibiotic sensitive microorganisms involved in infectious keratitis.

BACKGROUND: Corneal infections with antibiotic-resistant microorganisms are an increasingly difficult management challenge and chemically or photochemically cross-linking the cornea for therapy presents a unique approach to managing such infections since both direct microbial pathogens killing and matrix stabilization can occur simultaneously. The present study was undertaken in order to compare the anti-microbial efficacy, in vitro, of 5 candidate cross-linking solutions against 5 different microbial pathogens with relevance to infectious keratitis. METHODS: In vitro bactericidal efficacy studies were carried out using 5 different FARs [diazolidinyl urea (DAU), 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM), sodium hydroxymethylglycinate (SMG), 2-(hydroxymethyl)-2-nitro-1,3-propanediol (NT = nitrotriol), 2-nitro-1-propanol (NP)] against 5 different microbial pathogens including two antibiotic-resistant species [methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Pseudomonas aeruginosa (PA), and Candida albicans (CA)]. Standard in vitro antimicrobial testing methods were used. RESULTS: The results for MSSA were similar to those for MRSA. DAU, DMDM, and SMG all showed effectiveness with greater effects generally observed with longer incubation times and higher concentrations. Against MRSA, 40 mM SMG at 120 min showed a > 95% kill rate, p < 0.02. Against VRE, 40 mM DAU for 120 min showed a > 94% kill rate, p < 0.001. All FARs showed bactericidal effect against Pseudomonas aeruginosa, making PA the most susceptible of the strains tested. Candida showed relative resistance to these compounds, requiring high concentrations (100 mM) to achieve kill rates greater than 50%. CONCLUSION: Our results show that each FAR compound has different effects against different cultures. Our antimicrobial armamentarium could potentially be broadened by DAU, DMDM, SMG and other FARs for antibiotic-resistant keratitis. Further testing in live animal models are indicated.

Identification of benzo[d]pyrrolo[2,1-b]thiazole derivatives as CENP-E inhibitors.

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[d]pyrrolo[2,1-b]thiazole derivatives, including 1, as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins.

Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids.

Stimuli-responsive polymersomes formed by amphiphilic block copolymers have attracted substantial attention as smart and robust containers for drug delivery and nano/microreactors. Biosourced amphiphilic diblock copolypeptoids were developed that can self-assemble into oxidation-responsive unilamellar vesicles. These vesicles can burst under the action of reactive oxygen species which can be the hydrogen peroxide or the singlet oxygen produced by light-activation of a photosensitizer with spatiotemporal control. Polysarcosine (PSar, also called poly(N-methyl glycine)) was selected as the hydrophilic block because of its resistance to protein adsorption and low toxicity, similar to poly(ethylene glycol) (PEG). We designed and synthesized poly(N-3-(methylthio)propyl glycine) as the hydrophobic block. Its polyglycine backbone is the same as that of PSar, and especially, its hydrophobic N-substituents, thioether side chains, can be oxidized to hydrophilic sulfoxides. These oxidation-responsive polymersomes entirely based on N-substituted poly(amino acid)s were biocompatible as confirmed by cell viability tests and may find applications in drug delivery, biosensing, biodetection, and nano/microreactors.

The impact of D-cycloserine and sarcosine on in vivo frontal neural activity in a schizophrenia-like model.

BACKGROUND: N-methyl-D-aspartate receptor (NMDAR) hypofunction has been proposed to underlie the pathogenesis of schizophrenia. Specifically, reduced function of NMDARs leads to altered balance between excitation and inhibition which further drives neural network malfunctions. Clinical studies suggested that NMDAR modulators (glycine, D-serine, D-cycloserine and glycine transporter inhibitors) may be beneficial in treating schizophrenia patients. Preclinical evidence also suggested that these NMDAR modulators may enhance synaptic NMDAR function and synaptic plasticity in brain slices. However, an important issue that has not been addressed is whether these NMDAR modulators modulate neural activity/spiking in vivo. METHODS: By using in vivo calcium imaging and single unit recording, we tested the effect of D-cycloserine, sarcosine (glycine transporter 1 inhibitor) and glycine, on schizophrenia-like model mice. RESULTS: In vivo neural activity is significantly higher in the schizophrenia-like model mice, compared to control mice. D-cycloserine and sarcosine showed no significant effect on neural activity in the schizophrenia-like model mice. Glycine induced a large reduction in movement in home cage and reduced in vivo brain activity in control mice which prevented further analysis of its effect in schizophrenia-like model mice. CONCLUSIONS: We conclude that there is no significant impact of the tested NMDAR modulators on neural spiking in the schizophrenia-like model mice.

Dysregulated Glycine Signaling Contributes to Increased Impulsivity during Protracted Alcohol Abstinence.

Persons with alcoholism who are abstinent exhibit persistent impairments in the capacity for response inhibition, and this form of impulsivity is significantly associated with heightened relapse risk. Brain-imaging studies implicate aberrant prefrontal cortical function in this behavioral pathology, although the underlying mechanisms are not understood. Here we present evidence that deficient activation of glycine and serine release in the ventral medial prefrontal cortex (vmPFC) contributes to increased motor impulsivity during protracted abstinence from long-term alcohol exposure. Levels of 12 neurotransmitters were monitored in the rat vmPFC during the performance of a challenging variant of the five-choice serial reaction time task (5-CSRTT) in which alcohol-exposed rats exhibit excessive premature responding. Following long-term ethanol exposure, rats showed blunted task-related recruitment of vmPFC glycine and serine release, and the loss of an inverse relationship between levels of these neurotransmitters and premature responding normally evident in alcohol-naive subjects. Intra-vmPFC administration of the glycine transport inhibitor ALX5407 prevented excessive premature responding by alcohol-exposed rats, and this was reliant on NMDA glycine site availability. Alcohol-exposed rats and controls did not differ in their premature responding and glycine and serine levels in vmPFC during the performance of the standard 5-CSRTT. Collectively, these findings provide novel insight into cortical neurochemical mechanisms contributing to increased impulsivity following long-term alcohol exposure and highlight the NMDA receptor coagonist site as a potential therapeutic target for increased impulsivity that may contribute to relapse risk.SIGNIFICANCE STATEMENT Persons with alcoholism demonstrate increased motor impulsivity during abstinence; however, the neuronal mechanisms underlying these behavioral effects remain unknown. Here, we took advantage of an animal model that shows deficiencies in inhibitory control following prolonged alcohol exposure to investigate the neurotransmitters that are potentially responsible for dysregulated motor impulsivity following long-term alcohol exposure. We found that increased motor impulsivity is associated with reduced recruitment of glycine and serine neurotransmitters in the ventromedial prefrontal cortex (vmPFC) cortex in rats following long-term alcohol exposure. Administration of glycine transport inhibitor ALX5407 in the vmPFC alleviated deficits in impulse control.

Flagellar-associated Protein FAP85 Is a Microtubule Inner Protein That Stabilizes Microtubules.

Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing ß-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubule-binding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.Key words: Chlamydomonas, flagella, doublet microtubule, microtubule inner proteins.

MSA prions exhibit remarkable stability and resistance to inactivation.

In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83+/-), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/- mice developed neurological signs accompanied by α-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate α-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/- mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of α-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.

MPT0B169 and MPT0B002, New Tubulin Inhibitors, Induce Growth Inhibition, G2/M Cell Cycle Arrest, and Apoptosis in Human Colorectal Cancer Cells.

We previously synthesized new tubulin inhibitors, MPT0B169 and MPT0B002, which induced growth inhibition and apoptosis in leukemia cells. However, their effects on solid tumor cells have not been determined. In this study, we investigated the effects of MPT0B169 and MPT0B002 on glioblastoma, breast, lung, and colorectal cancer (CRC) cell lines. A cell viability analysis showed that MPT0B169 and MPT0B002 were more effective in inhibiting the proliferation of COLO205 and HT29 CRC cells than U87MG and GBM8401 glioblastoma, MCF-7 and MDA-MB-231 breast cancer, and A549 lung cancer cells. MPT0B169 and MPT0B002 inhibited growth of COLO205 and HT29 cells in dose- and time-dependent manners. A colony-formation assay confirmed the growth inhibitory effects of MPT0B169 and MPT0B002 on COLO205 and HT29 cells. MPT0B169 and MPT0B002 disrupted tubulin polymerization and arrested the cell cycle at the G2/M phase, with a concomitant increase of the cyclin B1 level. MPT0B169 and MPT0B002 induced apoptosis, accompanied by induction of the intrinsic apoptotic pathway, as shown by a reduction in the caspase-9 level and increases in cleaved caspase-3 and cleaved PARP. These results suggest that MPT0B169 and MPT0B002, new tubulin inhibitors, induced growth inhibition, G2/M arrest, and apoptosis in COLO205 and HT29 cells, and they could potentially be anticancer agents for CRC cells.

Differential regulation of NMDA receptors by d-serine and glycine in mammalian spinal locomotor networks.

Activation of N-methyl-d-aspartate receptors (NMDARs) requires the binding of a coagonist, either d-serine or glycine, in addition to glutamate. Changes in occupancy of the coagonist binding site are proposed to modulate neural networks including those controlling swimming in frog tadpoles. Here, we characterize regulation of the NMDAR coagonist binding site in mammalian spinal locomotor networks. Blockade of NMDARs by d(-)-2-amino-5-phosphonopentanoic acid (d-APV) or 5,7-dichlorokynurenic acid reduced the frequency and amplitude of pharmacologically induced locomotor-related activity recorded from the ventral roots of spinal-cord preparations from neonatal mice. Furthermore, d-APV abolished synchronous activity induced by blockade of inhibitory transmission. These results demonstrate an important role for NMDARs in murine locomotor networks. Bath-applied d-serine enhanced the frequency of locomotor-related but not disinhibited bursting, indicating that coagonist binding sites are saturated during the latter but not the former mode of activity. Depletion of endogenous d-serine by d-amino acid oxidase or the serine-racemase inhibitor erythro-ß-hydroxy-l-aspartic acid (HOAsp) increased the frequency of locomotor-related activity, whereas application of l-serine to enhance endogenous d-serine synthesis reduced burst frequency, suggesting a requirement for d-serine at a subset of synapses onto inhibitory interneurons. Consistent with this, HOAsp was ineffective during disinhibited activity. Bath-applied glycine (1-100 µM) failed to alter locomotor-related activity, whereas ALX 5407, a selective inhibitor of glycine transporter-1 (GlyT1), enhanced burst frequency, supporting a role for GlyT1 in NMDAR regulation. Together these findings indicate activity-dependent and synapse-specific regulation of the coagonist binding site within spinal locomotor networks, illustrating the importance of NMDAR regulation in shaping motor output.NEW & NOTEWORTHY We provide evidence that NMDARs within murine spinal locomotor networks determine the frequency and amplitude of ongoing locomotor-related activity in vitro and that NMDARs are regulated by d-serine and glycine in a synapse-specific and activity-dependent manner. In addition, glycine transporter-1 is shown to be an important regulator of NMDARs during locomotor-related activity. These results show how excitatory transmission can be tuned to diversify the output repertoire of spinal locomotor networks in mammals.

Effects of sarcosine and N, N-dimethylglycine on NMDA receptor-mediated excitatory field potentials.

BACKGROUND: Sarcosine, a glycine transporter type 1 inhibitor and an N-methyl-D-aspartate (NMDA) receptor co-agonist at the glycine binding site, potentiates NMDA receptor function. Structurally similar to sarcosine, N,N-dimethylglycine (DMG) is also N-methyl glycine-derivative amino acid and commonly used as a dietary supplement. The present study compared the effects of sarcosine and DMG on NMDA receptor-mediated excitatory field potentials (EFPs) in mouse medial prefrontal cortex brain slices using a multi-electrode array system. RESULTS: Glycine, sarcosine and DMG alone did not alter the NMDA receptor-mediated EFPs, but in combination with glutamate, glycine and its N-methyl derivatives significantly increased the frequency and amplitude of EFPs. The enhancing effects of glycine analogs in combination with glutamate on EFPs were remarkably reduced by the glycine binding site antagonist 7-chlorokynurenate (7-CK). However, DMG, but not sarcosine, reduced the frequency and amplitude of EFPs elicited by co-application of glutamate plus glycine. D-cycloserine, a partial agonist at the glycine binding site on NMDA receptors, affected EFPs in a similar manner to DMG. Furthermore, DMG, but not sarcosine, reduced the frequencies and amplitudes of EFPs elicited by glutamate plus D-serine, another endogenous ligand for glycine binding site. CONCLUSIONS: These findings suggest that sarcosine acts as a full agonist, yet DMG is a partial agonist at glycine binding site of NMDA receptors. The molecular docking analysis indicated that the interactions of glycine, sarcosine, and DMG to NMDA receptors are highly similar, supporting that the glycine binding site of NMDA receptors is a critical target site for sarcosine and DMG.

Electrophysiological evidence of increased glycine receptor-mediated phasic and tonic inhibition by blockade of glycine transporters in spinal superficial dorsal horn neurons of adult mice.

To understand the synaptic and/or extrasynaptic mechanisms underlying pain relief by blockade of glycine transporter subtypes GlyT1 and GlyT2, whole-cell recordings were made from dorsal horn neurons in spinal slices from adult mice, and the effects of NFPS and ALX-1393, selective GlyT1 and GlyT2 inhibitors, respectively, on phasic evoked or miniature glycinergic inhibitory postsynaptic currents (eIPSCs or mIPSCs) were examined. NFPS and ALX-1393 prolonged the decay phase of eIPSCs without affecting their amplitude. In the presence of tetrodotoxin to record mIPSCs, NFPS and ALX-1393 induced a tonic inward current that was reversed by strychnine. Although NFPS had no statistically significant influences on mIPSCs, ALX-1393 significantly increased their frequency. We then further explored the role of GlyTs in the maintenance of glycinergic IPSCs. To facilitate vesicular release of glycine, repetitive high-frequency stimulation (HFS) was applied at 10 Hz for 3 min during continuous recordings of eIPSCs at 0.1 Hz. Prominent suppression of eIPSCs was evident after HFS in the presence of ALX-1393, but not NFPS. Thus, it appears that phasic and tonic inhibition may contribute to the analgesic effects of GlyT inhibitors. However, reduced glycinergic inhibition due to impaired vesicular refilling could hamper the analgesic efficacy of GlyT2 inhibitors.

Methamphetamine increases Prion Protein and induces dopamine-dependent expression of protease resistant PrPsc.

The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

GlyT1 Inhibitor NFPS Exerts Neuroprotection via GlyR Alpha1 Subunit in the Rat Model of Transient Focal Cerebral Ischaemia and Reperfusion.

BACKGROUND/AIMS: Glycine is a strychnine-sensitive inhibitory neurotransmitter in the central nervous system (CNS), especially in the spinal cord, brainstem, and retina. The objective of the present study was to investigate the potential neuroprotective effects of GlyT1 inhibitor N [3-(4'-fluorophenyl)-3-(4'-phenylphenoxy) propyl] sarcosine (NFPS) in the rat model of experimental stroke. METHODS: In vivo ischaemia was induced by transient middle cerebral artery occlusion (tMCAO). The methods of Western Blotting, Nissl Staining and Morris water maze methods were applied to analyze the anti-ischaemia mechanism. RESULTS: The results showed that high dose of NFPS (H-NFPS) significantly reduced infarct volume, neuronal injury and the expression of cleaved caspase-3, enhanced Bcl-2/Bax, and improved spatial learning deficits which were administered three hours after transient middle cerebral artery occlusion (tMCAO) induction in rats, while, low dose of NFPS (L-NFPS) exacerbated the injury of ischaemia. These findings suggested that low and high dose of NFPS produced opposite effects. Importantly, it was demonstrated that H-NFPS-dependent neuronal protection was inverted by salicylate (Sal), a specific GlyR x0251;1 antagonist. Such effects could probably be attributed to the enhanced glycine level in both synaptic and extrasynaptic clefts and the subsequently altered extrasynaptic GlyRs and their subtypes. CONCLUSIONS: These data imply that GlyT1 inhibitor NFPS may be a novel target for clinical treatment of transient focal cerebral ischaemia and reperfusion which are associated with altered GlyR alpha 1 subunits.

MPT0B169, a novel tubulin inhibitor, induces apoptosis in taxol-resistant acute myeloid leukemia cells through mitochondrial dysfunction and Mcl-1 downregulation.

Acute myeloid leukemia (AML) is a hematological malignant disorder. AML cells are not susceptible to chemotherapeutic drugs because of their multidrug resistance (MDR). Antitubulin agents are currently employed in cancer treatments; however, drug resistance results in treatment failures because of MDR1 expressing cancer cells. We previously synthesized a new tubulin inhibitor, 2-dimethylamino-N-[1-(4-methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-acetamide (MPT0B169), which inhibits AML cell proliferation by arresting cell cycle at the G2/M phase. In this study, we explored the effect of MPT0B169 on apoptosis in AML HL60 and NB4 cells and MDR1-mediated taxol-resistant HL60/TaxR cells and the underlying mechanism. MPT0B169 induced concentration- and time-dependent apoptosis in these cancer cells, as observed through annexin V/propidium iodide double staining and flow cytometry. Furthermore, DNA fragmentation analysis confirmed MPT0B169-induced apoptosis. MPT0B169 induced a loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, cleavage and activation of caspase-9 and caspase-3, and consequently cleavage of poly (ADP ribose) polymerase. Western blot analysis showed that MPT0B169 markedly reduced Mcl-1 (an antiapoptotic protein) levels; however, it caused no changes in Bcl-2 or BAX (a proapoptotic protein). Knockdown of Mcl-1 using small interfering RNA (siRNA) slightly induced growth inhibition and apoptosis in the HL60 and HL60/TaxR cells. Further investigation revealed that Mcl-1 siRNA enhanced the sensitivity of HL60 and HL60/TaxR cells to MPT0B169-induced growth inhibition and apoptosis. Together, these results demonstrated that MPT0B169-induced apoptosis in nonresistant and MDR1-mediated taxol-resistant AML cells through Mcl-1 downregulation and a mitochondria-mediated pathway. MPT0B169 can overcome MDR1-mediated drug resistance in AML cells.