Alpha-secretase inhibition impairs Group I metabotropic glutamate receptor-mediated protein synthesis, long-term potentiation and long-term depression.

Group I metabotropic glutamate receptors (Gp1-mGluRs) exert a host of effects on cellular functions, including enhancement of protein synthesis and the associated facilitation of long-term potentiation (LTP) and induction of long-term depression (LTD). However, the complete cascades of events mediating these events are not fully understood. Gp1-mGluRs trigger α-secretase cleavage of amyloid precursor protein, producing soluble amyloid precursor protein-α (sAPPα), a known regulator of LTP. However, the α-cleavage of APP has not previously been linked to Gp1-mGluR's actions. Using rat hippocampal slices, we found that the α-secretase inhibitor tumour necrosis factor-alpha protease inhibitor-1, which inhibits both disintegrin and metalloprotease 10 (ADAM10) and 17 (ADAM17) activity, blocked or reduced the ability of the Gp1-mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) to stimulate protein synthesis, metaplastically prime future LTP and elicit sub-maximal LTD. In contrast, the specific ADAM10 antagonist GI254023X did not affect the regulation of plasticity, suggesting that ADAM17 but not ADAM10 is involved in mediating these effects of DHPG. However, neither drug affected LTD that was strongly induced by either high-concentration DHPG or paired-pulse synaptic stimulation. Our data suggest that moderate Gp1-mGluR activation triggers α-secretase sheddase activity targeting APP or other membrane-bound proteins as part of a more complex signalling cascade than previously envisioned. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Synergistic protective effects of 3,4-dihydroxyphenylglycol and hydroxytyrosol in male rats against induced heat stress-induced reproduction damage.

Testicular heat stress disrupts spermiogenesis and damages testicular tissue. The study aims to assess 3,4-dihydroxyphenylglycol (DHPG) and hydroxytyrosol (HT) from olive oil as antioxidants to reduce heat-induced testicular damage. Seven groups of 35 male rats were used. Group I got normal saline. Group 2 had HS (43 °C for 20 min/day) and normal saline for 60 days. Groups 3-7 had HS and DHPG/HT doses (0.5 mg/kg DHPG, 1 mg/kg DHPG, 5 mg/kg HT, 0.5 mg/kg DHPG + 5 mg/kg HT, and 1 mg/kg DHPG + 5 mg/kg HT). The evaluation included tests on testicular tissue, sperm quality, oxidative status, gene activity, and fertility after 60 days. After DHPG and HT treatment, sperm motility, viability, and plasma membrane functionality, as well as levels of total antioxidant capacity (TAC), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), and Bcl-2 gene expression, and in vivo fertility indexes increased. Meanwhile, abnormal morphology and DNA damage decreased, along with levels of glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA), and Bax, caspase-3, and caspase-9 gene expression, compared to the HS group. The study found that DHPG and HT have a more substantial synergistic effect when used together, improving reproductive health.

mGluR1/IP3/ERK signaling pathway regulates vestibular compensation in ON UBCs of the cerebellar flocculus.

AIMS: To investigate the role of mGluR1α in cerebellar unipolar brush cells (UBC) in mediating vestibular compensation (VC), using mGluR1α agonist and antagonist to modulate ON UBC neurons, and explore the mGluR1/IP3/extracellular signal-regulated kinase (ERK) signaling pathway. METHODS: First, AAV virus that knockdown ON UBC (mGluR1α) were injected into cerebellar UBC by stereotactic, and verified by immunofluorescence and western blot. The effect on VC was evaluated after unilateral labyrinthectomy (UL). Second, saline, (RS)-3,5-dihydroxyphenylglycine (DHPG), and LY367385 were injected into tubes implanted in rats at different time points after UL separately. The effect on ON UBC neuron activity was evaluated by immunofluorescence. Then, Phosphoinositide (PI) and p-ERK1/2 levels of mGluR1α were analyzed by ELISA after UL. The protein levels of p-ERK and total ERK were verified by western blot. In addition, the effect of mGluR1α activation or inhibition on VC-related behavior was observed. RESULTS: mGluR1α knockdown induced VC phenotypes. DHPG increased ON UBC activity, while LY367385 reduced ON UBC activity. DHPG group showed an increase in PI and p-ERK1/2 levels, while LY367385 group showed a decrease in PI and p-ERK1/2 levels in cerebellar UBC of rats. The western blot results of p-ERK and total ERK confirm and support the observations. DHPG alleviated VC-related behavior phenotypes, while LY367385 exacerbated vestibular decompensation-like behavior induced by UL. CONCLUSION: mGluR1α activity in cerebellar ON UBC is crucial for mediating VC through the mGluR1/IP3/ERK signaling pathway, which affects ON UBC neuron activity and contributes to the pathogenesis of VC.

[Effect of Rehmanniae Radix on depression-like behavior and hippocampal monoamine neurotransmitters of chronic unpredictable mild stress model rats].

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.

Synaptic protein interaction networks encode experience by assuming stimulus-specific and brain-region-specific states.

A core network of widely expressed proteins within the glutamatergic post-synapse mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we address the question, how does proteomic composition affect activity-dependent protein-protein interaction networks (PINs) downstream of synaptic activity? Using quantitative multiplex co-immunoprecipitation, we compare the PIN response of in vivo or ex vivo neurons derived from different brain regions to activation by different agonists or different forms of eyeblink conditioning. We report that PINs discriminate between incoming stimuli using differential kinetics of overlapping and non-overlapping PIN parameters. Further, these "molecular logic rules" differ by brain region. We conclude that although the PIN of the glutamatergic post-synapse is expressed widely throughout the brain, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity. This diversity may help explain the challenges in developing molecule-specific drug therapies for neurological disorders.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Reelin Regulates Neuronal Excitability through Striatal-Enriched Protein Tyrosine Phosphatase (STEP61) and Calcium Permeable AMPARs in an NMDAR-Dependent Manner.

Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the accumulation of amyloid-ß (Aß) plaques and neurofibrillary tangles. Aß oligomers cause synaptic dysfunction early in AD by enhancing long-term depression (LTD; a paradigm for forgetfulness) via metabotropic glutamate receptor (mGluR)-dependent regulation of striatal-enriched tyrosine phosphatase (STEP61). Reelin is a neuromodulator that signals through ApoE (apolipoprotein E) receptors to protect the synapse against Aß toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aß-oligomers activate metabotropic glutamate receptors (Renner et al., 2010). We therefore asked whether Reelin might also affect mGluR-LTD. To this end, we induced chemical mGluR-LTD using DHPG (Dihydroxyphenylglycine), a selective mGluR5 agonist. We found that exogenous Reelin reduces the DHPG-induced increase in STEP61, prevents the dephosphorylation of GluA2, and concomitantly blocks mGluR-mediated LTD. By contrast, Reelin deficiency increased expression of Ca2+-permeable GluA2-lacking AMPA receptors along with higher STEP61 levels, resulting in occlusion of DHPG-induced LTD in hippocampal CA1 neurons. We propose a model in which Reelin modulates local protein synthesis as well as AMPA receptor subunit composition through modulation of mGluR-mediated signaling with implications for memory consolidation or neurodegeneration.SIGNIFICANCE STATEMENT Reelin is an important neuromodulator, which in the adult brain controls synaptic plasticity and protects against neurodegeneration. Amyloid-ß has been shown to use mGluRs to induce synaptic depression through endocytosis of NMDA and AMPA receptors, a mechanism referred to as LTD, a paradigm of forgetfulness. Our results show that Reelin regulates the phosphatase STEP, which plays an important role in neurodegeneration, as well as the expression of calcium-permeable AMPA receptors, which play a role in memory formation. These data suggest that Reelin uses mGluR LTD pathways to regulate memory formation as well as neurodegeneration.

Differential abnormalities of cerebrospinal fluid dopaminergic versus noradrenergic indices in synucleinopathies.

The synucleinopathies Parkinson's disease (PD), multiple system atrophy (MSA), and pure autonomic failure (PAF) are characterized by intra-cytoplasmic deposition of the protein alpha-synuclein and by catecholamine depletion. PAF, which manifests with neurogenic orthostatic hypotension (nOH) and no motor signs of central neurodegeneration, can evolve into PD+nOH. The cerebrospinal fluid (CSF) levels of catecholamine metabolites may indicate central catecholamine deficiency in these synucleinopathies, but the literature is inconsistent and incomplete. In this retrospective cohort study we reviewed data about CSF catecholamines, the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and the norepinephrine metabolites 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG). The compounds were measured in 36 patients with PD, 37 patients with MSA, and 19 patients with PAF and in 38 controls. Compared to the control group, the PD, MSA, and PAF groups had decreased CSF MHPG (p < .0001 each by Dunnett's post hoc test), DHPG (p = .004; p < .0001; p < .0001) and norepinephrine (p = .017; p = .0003; p = .044). CSF HVA and DOPAC were decreased in PD (p < .0001 each) and MSA (p < .0001 each) but not in PAF. The three synucleinopathies therefore have in common in vivo evidence of central noradrenergic deficiency but differ in the extents of central dopaminergic deficiency-prominent in PD and MSA, less apparent in PAF. Data from putamen 18 F-DOPA and cardiac 18 F-dopamine neuroimaging in the same patients, post-mortem tissue catecholamines in largely separate cohorts, and review of the neuropathology literature fit with these distinctions. The results suggest a 'norepinephrine first' ascending pathogenetic sequence in synucleinopathies, with degeneration of pontine locus ceruleus noradrenergic neurons preceding the loss of midbrain substantia nigra dopaminergic neurons.

Regulation of mGluR1 on the Expression of PKC and NMDAR in Aluminum-Exposed PC12 Cells.

Aluminum demonstrates clear neurotoxicity and can cause Alzheimer's disease (AD)-like symptoms, including cognitive impairment. One toxic effect of aluminum is a decrease in synaptic plasticity, but the specific mechanism remains unclear. In this study, PC12 cells were treated with Al(mal)3 to construct a toxic cell model. (S)-3,5-Dihydroxyphenylglycine (DHPG), α-methyl-4-carboxyphenylglycine (MCPG), and mGluR1-siRNA were used to interfere with the expression of metabotropic glutamate receptor subtype 1 (mGluR1). Polymerase chain reaction and western blotting were used to investigate the expression of mGluR1, protein kinase C (PKC), and N-methyl-D-aspartate receptor (NMDAR) subunits. ELISA was used to detect PKC enzyme activity. In PC12 cells, mRNA and protein expressions of PKC and NMDAR subunits were inhibited by Al(mal)3. Aluminum may further regulate the expression of NMDAR1 and NMDAR2B through mGluR1 to regulate PKC enzyme activity, thereby affecting learning and memory functions. Furthermore, the results implied that the mGluR1-PKC-NMDAR signaling pathway may predominately involve positive regulation. These findings provide new targets for studying the neurotoxic mechanism of aluminum.

Ribosome profiling in mouse hippocampus: plasticity-induced regulation and bidirectional control by TSC2 and FMRP.

BACKGROUND: Mutations in TSC2 are the most common cause of tuberous sclerosis (TSC), a disorder with a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD) and behavior, but the identity of mRNAs responsive to mGluR-LTD signaling is largely unknown. METHODS: We utilized Tsc2+/- mice as a mouse model of TSC and prepared hippocampal slices from these animals. We induced mGluR-LTD synaptic plasticity in slices and processed the samples for RNA-seq and ribosome profiling to identify differentially expressed genes in Tsc2+/- and following mGluR-LTD synaptic plasticity. RESULTS: Ribosome profiling reveals that in Tsc2+/- mouse hippocampal slices, the expression of several mRNAs was dysregulated: terminal oligopyrimidine (TOP)-containing mRNAs decreased, while FMRP-binding targets increased. Remarkably, we observed the opposite changes of FMRP binding targets in Fmr1-/y hippocampi. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady-state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in TSC mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in TSC mice. CONCLUSION: These results suggest a molecular basis for bidirectional regulation of synaptic plasticity and behavior by TSC2 and FMRP. Our study also suggests that altered mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/- mice.

Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

Endocannabinoid receptors contribute significantly to multiple forms of long-term depression in the rat dentate gyrus.

Cannabinoid receptors are widely expressed throughout the hippocampal formation, but are particularly dense in the dentate gyrus (DG) subregion. We, and others, have shown in mice that cannabinoid type 1 receptors (CB1Rs) are involved in a long-term depression (LTD) that can be induced by prolonged 10 Hz stimulation of the medial perforant path (MPP)-granule cell synaptic input to the DG. Here, we extend this work to examine the involvement of CB1Rs in other common forms of LTD in the hippocampus of juvenile male and female Sprague-Dawley rats (Rattus norvegicus). We found, as in mice, that prolonged 10 Hz stimulation (6000 pulses) could reliably induce a form of LTD that was dependent upon CB1R activation. In addition, we also discovered a role for both CB1R and mGluR proteins in LTD induced with 1 Hz low-frequency stimulation (1 Hz-LTD; 900 pulses) and in LTD induced by bath application of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG; DHPG-LTD). This study elucidates an essential role for endocannabinoid receptors in a number of forms of LTD in the rat DG, and identifies a novel role for CB1Rs as potential therapeutic targets for conditions that involve impaired LTD in the DG.

Tumor necrosis factor-alpha aggravates gliosis and inflammation of activated retinal Müller cells.

Tumor necrosis factor-alpha (TNF-α), a major inflammatory factor released from activated retinal glial cells, is implicated in the pathogenesis of glaucoma. In this study, we investigated whether and how TNF-α may affect functional conditions of activated retinal Müller cells. Our results showed that in the group I metabotropic glutamate receptor (mGluR I) agonist DHPG-activated cultured Müller cells, TNF-α treatment aggravated cell gliosis, as evidenced by significantly increased expression of glial fibrillary acidic protein (GFAP). TNF-α treatment of the DHPG-activated Müller cells decreased cell proliferation and induced cell apoptosis. In normal Müller cells, TNF-α treatment increased the mRNA levels of leukocyte inhibitory factor (LIF), intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and chemokine C-C-motif ligand 2 (CCL2), which could be significantly attenuated when Müller cells were pre-activated. However, TNF-α-induced elevation in mRNA levels of inflammatory factors, such as TNF-α, inducible nitric oxide synthase (iNOS), and interleukin-6 (IL-6), in normal Müller cells still kept higher levels when Müller cells were pre-activated. Furthermore, the TNF-α-induced changes of cytokines were partially mediated by NF-κB signaling pathway. Our results suggest that TNF-α may promote gliosis and inflammatory response of activated Müller cells, thus aggravating RGC injury in glaucoma.

Differential contribution of metabotropic glutamate receptor 5 common allosteric binding site residues to biased allosteric agonism.

Allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) represent an attractive therapeutic strategy for multiple CNS disorders. Chemically distinct mGlu5 positive allosteric modulators (PAMs) that interact with a common binding site can demonstrate biased allosteric agonism relative to the orthosteric agonist, DHPG, when comparing activity in signaling assays such as IP1 accumulation, ERK1/2 phosphorylation (pERK1/2) and iCa2+ mobilization. However, the structural basis for such biased agonism is not well understood. Therefore, we evaluated biased allosteric agonism mediated by four mGlu5 PAM-agonists from diverse chemical scaffolds (i.e., DPFE, VU0409551, VU29, and VU0424465) for three measures of mGlu5 activation (i.e., iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation) at eight single-point mutations within the common allosteric binding pocket of mGlu5. In particular, mGlu5 allosteric site mutations had differential effects on the intrinsic efficacy of mGlu5 PAMs for multiple signaling pathways. Specifically, a loss of agonism for iCa2+ mobilization was evident for DPFE and VU0409551 for most mutants, whereas IP1 accumulation and ERK phosphorylation were retained, albeit with reduced maximal responses. Additionally, bias profiles between iCa2+ mobilization and IP1/ERK pathways remained similar to wild type for most mutants. However, W784A and A809G mutants lost bias between IP1 accumulation and ERK phosphorylation for VU0424465, whereas a loss of bias between iCa2+ mobilization and ERK1/2 phosphorylation was evident for F787A, S808A and A809G mutants. These data provide further insight into the structural requirements for allosteric agonism across multiple mGlu5-mediated signaling pathways. An understanding of mGlu5 biased agonism at a structural level may provide the foundation for rational structure-based design of biased allosteric ligands for the treatment of neurological disorders.

Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site.

The metabotropic glutamate receptor 5 (mGlu5) is a promising therapeutic target for multiple CNS disorders. Recent mGlu5 drug discovery has focused on targeting binding sites within the mGlu5 7-transmembrane domain (7TM) that are topographically distinct from that of the endogenous ligand. mGlu5 primarily couples to Gq/11 proteins leading to mobilization of intracellular Ca2+ (iCa2+), but also activates iCa2+ independent signaling pathways, with biased agonism/modulation operative for multiple positive allosteric modulator (PAM) and PAM-agonist chemotypes. Although several residues within the common allosteric binding pocket are key determinants of PAM activity, how these residues affect biased modulation is unknown. The current study probed the molecular basis of mGlu5 PAM biased modulation. Modulation of mGlu5 activity by four chemically distinct mGlu5 PAMs (VU0424465, DPFE, VU29 and VU0409551) was assessed across two distinct receptor endpoints (iCa2+ mobilization and ERK1/2 phosphorylation) at mGlu5 receptors containing single-point mutations of allosteric binding pocket residues informed by computational modeling. Many mutations had differential effects on PAM affinity and cooperativity across signaling endpoints, resulting in gain or reversal of bias at the level of both affinity and functional cooperativity. Additionally, mutants had differential effects on functional cooperativity between the orthosteric ligands, DHPG and glutamate, and the PAMs, DPFE and VU29, but not VU0409551, indicating that probe dependence is linked to orthosteric agonists conferring activation states that differentially influence allosteric ligand-receptor interactions in a chemotype dependent fashion. Collectively, these data provide crucial insight into the residues that govern different activation states adopted by mGlu5 in order to signal via distinct intracellular pathways when co-bound by orthosteric agonists and PAMs.

Deciphering Biosynthetic Enzymes Leading to 4-Chloro-6-Methyl-5,7-Dihydroxyphenylglycine, a Non-Proteinogenic Amino Acid in Totopotensamides.

Totopotensamide A (TPM A, 1) is a polyketide-peptide glycoside featuring a nonproteinogenic amino acid 4-chloro-6-methyl-5,7-dihydroxyphenylglycine (ClMeDPG). The biosynthetic gene cluster (BGC) of totopotensamides (tot) was previously activated by manipulating transcription regulators in marine-derived Streptomyces pactum SCSIO 02999. Herein, we report the heterologous expression of the tot BGC in Streptomyces lividans TK64, and the production improvement of TPM A via in-frame deletion of two negative regulators totR5 and totR3. The formation of ClMeDPG was proposed to require six enzymes, including four enzymes TotC1C2C3C4 for 3,5-dihydroxyphenylglycine (DPG) biosynthesis and two modifying enzymes TotH (halogenase) and TotM (methyltransferase). Heterologous expression of the four-gene cassette totC1C2C3C4 led to production of 3,5-dihydroxyphenylglyoxylate (DPGX). The aminotransferase TotC4 was biochemically characterized to convert DPGX to S-DPG. Inactivation of totH led to a mutant accumulated a deschloro derivative TPM H1, and the ΔtotHi/ΔtotMi double mutant afforded two deschloro-desmethyl products TPMs HM1 and HM2. A hydrolysis experiment demonstrated that the DPG moiety in TPM HM2 was S-DPG, consistent with that of the TotC4 enzymatic product. These results confirmed that TotH and TotM were responsible for ClMeDPG biosynthesis. Bioinformatics analysis indicated that both TotH and TotM might act on thiolation domain-tethered substrates. This study provided evidence for deciphering enzymes leading to ClMeDPG in TPM A, and unambiguously determined its absolute configuration as S.

TRPC1 Regulates the Activity of a Voltage-Dependent Nonselective Cation Current in Hippocampal CA1 Neurons.

The cation channel subunit TRPC1 is strongly expressed in central neurons including neurons in the CA1 region of the hippocampus where it forms complexes with TRPC4 and TRPC5. To investigate the functional role of TRPC1 in these neurons and in channel function, we compared current responses to group I metabotropic glutamate receptor (mGluR I) activation and looked for major differences in dendritic morphology in neurons from TRPC1+/+ and TRPC1-/- mice. mGluR I stimulation resulted in the activation of a voltage-dependent nonselective cation current in both genotypes. Deletion of TRPC1 resulted in a modification of the shape of the current-voltage relationship, leading to an inward current increase. In current clamp recordings, the percentage of neurons that responded to depolarization in the presence of an mGluR I agonist with a plateau potential was increased in TRPC1-/- mice. There was also a small increase in the minor population of CA1 neurons that have more than one apical dendrite in TRPC1-/- mice. We conclude that TRPC1 has an inhibitory effect on receptor-operated nonselective cation channels in hippocampal CA1 neurons probably as a result of heterotetramer formation with other TRPC isoforms, and that TRPC1 deletion has only minor effects on dendritic morphology.

Effect of olive-derived antioxidants (3,4-dihydroxyphenylethanol and 3,4 dihydroxyphenylglycol) on sperm motility and fertility in liquid ram sperm stored at 15°C or 5°C.

The aim of the present study was to evaluate the effect of the addition of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during liquid storage at 5°C and 15°C. Semen was collected, pooled, diluted and then divided into aliquots supplemented with different concentrations (5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. Sperm motility characteristics were assessed in the different samples at 0, 6, 24, 48, 72 and 96 hr after cooling, and a fertility trial was also conducted. The results showed that the antioxidant addition did not significantly improve total and progressive motility in ovine cooled sperm maintained at 15° or 5°C. However, in samples stored at 5°C, LIN (48, 72, 96 hr), STR (0 hr) and WOB (0, 48, 72, 96 hr) values significantly decreased in comparison with control treatment when high antioxidant concentrations were added (MIX100 or HT100). When samples were maintained at 15°C, MIX50 showed significantly higher VCL values than the control treatment after 6 hr cooling, and MIX100 showed significantly lower VCL values at 96 hr after cooling. According to the artificial insemination trial, no significant differences were observed when antioxidants were added. In conclusion, the use of HT and DHPG showed small impact in sperm motility and fertility was not affected (nor detrimentally nor positively) when insemination was carried out using antioxidant-supplemented liquid sperm.

Tolerance Induced by (S)-3,5-Dihydroxyphenylglycine Postconditioning is Mediated by the PI3K/Akt/GSK3ß Signalling Pathway in an In Vitro Model of Cerebral Ischemia.

Ischemic postconditioning (PostC) is an endogenous neuroprotective strategy for cerebral ischemia induced by low activation of glutamate receptors. We have previously shown that the application of the mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) 5 min after 30 min of oxygen and glucose deprivation (OGD) reduces CA1 damage in organotypic hippocampal slices by activating the PI3K-Akt signalling pathway. In order to extend these data, we analysed the production of reactive oxygen species (ROS) and the glycogen synthase kinase 3ß (GSK3ß) signalling pathway. Our results show that DHPG PostC was associated with a reduction in the formation of ROS that is massively increased 24 h after OGD exposure. This reduction was prevented by the PI3K inhibitor LY294002, indicating that there is a link between the PI3K/Akt pathway and the formation of ROS in the protective mechanisms of PostC. DHPG PostC also induces a transient increased in GSK3ß phosphorylation and inactivation that is followed by nuclear accumulation of ß-catenin, that probably lead to the up-regulation of neuroprotective genes. Our results propose GSK3ß as new target for neuroprotection, therefore, we verified that the two GSK3ß inhibitors N-(3-Chloro-4-methylphenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-2-amine (TC-G 24) and LiCl are neuroprotective agents in OGD and also can be used as PostC agents.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.