Phorbol Ester Research Articles

Neuroligin-1 mediates presynaptic maturation through brain-derived neurotrophic factor signaling

BackgroundMaturation is a process that allows synapses to acquire full functionality, optimizing their activity to diverse neural circuits, and defects in synaptic maturation may contribute to neurodevelopmental disorders. Neuroligin-1 (NL1) is a postsynaptic cell adhesion molecule essential for synapse maturation, a role typically attributed to binding to pre-synaptic ligands, the neurexins. However, the pathways underlying the action of NL1 in synaptic maturation are incompletely understood, and some of its previously observed effects seem reminiscent of those described for the neurotrophin brain-derived neurotrophic factor (BDNF). Here, we show that maturational increases in active zone stability and synaptic vesicle recycling rely on the joint action of NL1 and brain-derived neurotrophic factor (BDNF).ResultsApplying BDNF to hippocampal neurons in primary cultures or organotypical slice cultures mimicked the effects of overexpressing NL1 on both structural and functional maturation. Overexpressing a NL1 mutant deficient in neurexin binding still induced presynaptic maturation. Like NL1, BDNF increased synaptic vesicle recycling and the augmentation of transmitter release by phorbol esters, both hallmarks of presynaptic maturation. Mimicking the effects of NL1, BDNF also increased the half-life of the active zone marker bassoon at synapses, reflecting increased active zone stability. Overexpressing NL1 increased the expression and synaptic accumulation of BDNF. Inhibiting BDNF signaling pharmacologically or genetically prevented the effects of NL1 on presynaptic maturation. Applying BDNF to NL1-knockout mouse cultures rescued defective presynaptic maturation, indicating that BDNF acts downstream of NL1 and can restore presynaptic maturation at late stages of network development.ConclusionsOur data introduce BDNF as a novel and essential component in a transsynaptic pathway linking NL1-mediated cell adhesion, neurotrophin action, and presynaptic maturation. Our findings connect synaptic cell adhesion and neurotrophin signaling and may provide a therapeutic approach to neurodevelopmental disorders by targeting synapse maturation.

Oxidative Stress and Multi-Organel Damage Induced by Two Novel Phytocannabinoids, CBDB and CBDP, in Breast Cancer Cells.

Over the last few years, much attention has been paid to phytocannabinoids derived from Cannabis for their therapeutic potential. Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the most abundant compounds of the Cannabis sativa L. plant. Recently, novel phytocannabinoids, such as cannabidibutol (CBDB) and cannabidiphorol (CBDP), have been discovered. These new molecules exhibit the same terpenophenolic core of CBD and differ only for the length of the alkyl side chain. Roles of CBD homologs in physiological and pathological processes are emerging but the exact molecular mechanisms remain to be fully elucidated. Here, we investigated the biological effects of the newly discovered CBDB or CBDP, compared to the well-known natural and synthetic CBD (nat CBD and syn CBD) in human breast carcinoma cells that express CB receptors. In detail, our data demonstrated that the treatment of cells with the novel phytocannabinoids affects cell viability, increases the production of reactive oxygen species (ROS) and activates cellular pathways related to ROS signaling, as already demonstrated for natural CBD. Moreover, we observed that the biological activity is significantly increased upon combining CBD homologs with drugs that inhibit the activity of enzymes involved in the metabolism of endocannabinoids, such as the monoacylglycerol lipase (MAGL) inhibitor, or with drugs that induces the activation of cellular stress pathways, such as the phorbol ester 12-myristate 13-acetate (PMA).

Benefits and Challenges of Jatropha Meal as Novel Biofeed for Animal Production.

Jatropha curcas L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing various bioactive compounds, including saponins, phytic acids, trypsin inhibitors, lectins, phenolics, and flavonoids, which render it as a potential biofeed for animal production. The Jatropha meal demonstrated various biological activities, including antioxidant, antibacterial, and anti-inflammatory effects which enhance its property as a bio-feed. The levels of these bioactive compounds in the seeds are dependent on the genotypes. The J. curcas possessed different varieties which are either toxic or non-toxic according to the presence of phorbol esters. The presence of phorbol esters in the meal confirmed the toxic variety of Jatropha resulting in the limited application of meal as a biofeed. The Jatropha meal devoid of phorbol esters could be applied as a biofeed in the animal production industry, and for the toxic varieties, various techniques such as physicochemical and biological treatments have been introduced to the industry to remove the phorbol esters from Jatropha meal. Several studies employing various cells and animals confirmed the toxicity of the phorbol esters. The molecular mechanism of action of phorbol esters is through up-regulation of PKC-β II gene, overexpression of down-stream proto-oncogenes resulted in inflammation and oxidative stress ending by apoptotic cell death. Despite the presence of valuable bioactive compounds in the Jatropha meal, its nutritional application is not recommended unless the phorbol esters are completely removed.

Single nucleotide polymorphism (SNP) of Jatropha curcas associated with the content of phorbol ester

Single nucleotide polymorphism (SNP) of Jatropha curcas associated with the content of phorbol ester

New phorbol ester derivatives as potent anti-HIV agents

Tigliane esters show many biological activities, including anti-HIV-1 activity. Our aim in this study was to establish structure-anti-HIV activity relationships for four series of tigliane-type diterpenoids. We synthesized and evaluated 29 new phorbol ester derivatives for anti-HIV activity and for cytotoxicity against human tumor cell lines. Among them, three derivatives, two phorbol-13-monoesters (5d and 5e) and a phorbol-12,13-diester (6a), showed significant anti-HIV activity. We found that better anti-HIV activity was often associated with a shorter acyl ester at C-13. Particularly, compounds with a phenyl ring in the ester side chain exhibited excellent anti-HIV activity and had good safety indexes. Due to its significant anti-HIV potency with a high selectivity index, phorbol-12,13-dicinnamoate (6a) was chosen as the potential candidate for further preclinical trials.

Multivariate Analysis of Seed Chemical Diversity among Jatropha curcas in Botswana

Jatropha (Jatropha curcas L.) has been identified as a potential bioenergy feedstock in arid regions, but knowledge of the diversity of its chemical characteristics is limited. In this study, 61 Jatropha accessions growing in Botswana, where both severe drought and winter frosts frequently occur, were analyzed for their seed chemical properties. Histogram analyses and meta-analysis comparisons with seeds from other countries/continents showed that the median/mean dry seed weight, toxic compound phorbol esters, and C18:0 fatty acid levels in the Botswanan accessions were lower than those from other countries/continents. A clustered heat map analysis indicated five clades for the Botswanan accessions, and their physicochemical traits were also categorized into five groups. Many positive and negative correlations were observed among the chemical traits, including negative correlations between the C18:3 (linolenic acid) content and yield-related traits (lipid content and dry seed weight). Principal component analysis highlighted the existence of accessions with highly deviated seed chemical compositions, such as those enriched in C18:0/C18:1 and C16:0/C16:1/C18:3 fatty acids. Overall, the present study suggests considerable diversity in the seed chemical compositions of Botswanan Jatropha accessions. Various accessions could be useful as feedstock for specific industrial products, as well as for breeding materials for the fortification of specific chemical ingredients.

Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells.

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.

Corneal Healing and Recovery of Ocular Crystallinity with a Dichloromethane Extract of Sedum dendroideum D.C. in a Novel Murine Model of Ocular Pterygium.

Pterygium is a corneal alteration that can cause visual impairment, which has been traditionally treated with the sap of Sedum dendroideum D.C. The pharmacological effect of a dichloromethane extract of S. dendroideum was demonstrated and implemented in a pterygium model on the healing process of corneal damage caused by phorbol esters. In mice of the ICR strain, a corneal lesion was caused by intravitreal injection of tetradecanoylphorbol acetate (TPA). The evolution of the corneal scarring process was monitored with vehicle, dexamethasone, and dichloromethane extract of S. dendroideum treatments by daily ophthalmic administration for fifteen days. The lesions were evaluated in situ with highlighted images of fluorescence of the lesions. Following treatment levels in eyeballs of IL-1α, TNF-α, and IL-10 cytokines were measured. The effective dose of TPA to produce a pterygium-like lesion was determined. The follow-up of the evolution of the scarring process allowed us to define that the treatment with S. dendroideum improved the experimental pterygium and had an immunomodulatory effect by decreasing TNF-α, IL-1α, and maintaining the level of IL-10 expression, without difference with respect to the healthy control. Traditional medical use of S. dendroideum sap to treat pterygium is fully justified by its compound composition.

Presynaptic Short-Term Plasticity Persists in the Absence of PKC Phosphorylation of Munc18-1.

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.

The Effect of Galangin on the Regulation of Vascular Contractility via the Holoenzyme Reactivation Suppressing ROCK/CPI-17 rather than PKC/CPI-17.

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

Quantification of Phorbol-12-myristate 13-acetate in Jatropha seed oil and cake at different stages of fruit maturity

ABSTRACT The current study investigates the influence of fruit maturity on Phorbol-12-myristate 13-acetate content in Jatropha seed oil as well as seed cake. Jatropha fruits used in this study were harvested from three different geographical locations in Botswana at four different fruit maturity stages, namely green yellow, yellow, yellow brown and brown dry. Phorbol esters in Jatropha seed oil and seed cake were quantified using High-Performance Liquid Chromatography. Results from this study reveal that phorbol ester content in Jatropha seeds varies with fruit maturity stage. Phorbol ester content in Jatropha seed oil and seed cake from yellow brown fruits had the highest value, hence was the most toxic. Phorbol-12-myristate 13-acetate content in Jatropha seed oil ranges from 3.4 to 4.2 mg/g whereas in seed cake it ranges from 1.7 to 2.0 mg/g during the four different fruit maturity stages investigated. Influence of geographical location on Phorbol-12-myristate 13-acetate content in Jatropha seeds is insignificant.

Endogenous neurosteroids pregnanolone and pregnanolone sulfate potentiate presynaptic glutamate release through distinct mechanisms.

Background and PurposeNeurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13‐dibutyrate (PDBu), a mimic of the second messenger DAG.Experimental ApproachWe use whole‐cell patch‐clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP‐tagged vesicle priming protein Munc13‐1, to explore the mechanisms of neurosteroid presynaptic modulation.Key ResultsPregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca2+ influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well‐known DAG/phorbol ester target Munc13‐1. However, TIRF microscopy experiments found no evidence of pregnanolone‐induced membrane translocation of GFP‐tagged Munc13‐1, suggesting that pregnanolone may regulate Munc13‐1 indirectly or interact with other DAG targets.Conclusion and ImplicationsWe describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca2+ influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.

CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Exploration of novel candidate genes involved in epidermal keratinocyte differentiation and skin barrier repair in man

A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect.In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.

Megakaryoblastic leukemia: a study on novel role of clinically significant long non-coding RNA signatures in megakaryocyte development during treatment with phorbol ester.

Acute megakaryocytic leukemia (AMKL) is one of the rarest sub-types of acute myeloid leukemia (AML). AMKL is characterized by high proliferation of megakaryoblasts and myelofibrosis of bone marrow, this disease is also associated with poor prognosis. Previous analyses have reported that the human megakaryoblastic cells can be differentiated into cells with megakaryocyte (MK)-like characteristics by phorbol 12-myristate 13-acetate (PMA). However, little is known about the mechanism responsible for regulating this differentiation process. We performed long non-coding RNA (lncRNA) profiling to investigate the differently expressed lncRNAs in megakaryocyte blast cells treated with and without PMA and examined those that may be responsible for the PMA-induced differentiation of megakaryoblasts into MKs. We found 30 out of 90 lncRNA signatures to be differentially expressed after PMA treatment of megakaryoblast cells, including the highly expressed JPX lncRNA. Further, in silico lncRNA-miRNA and miRNA-mRNA interaction analysis revealed that the JPX is likely involved in unblocking the expression of TGF-β receptor (TGF-βR) by sponging oncogenic miRNAs (miR-9-5p, miR-17-5p, and miR-106-5p) during MK differentiation. Further, we report the activation of TGF-βR-induced non-canonical ERK1/2 and PI3K/AKT pathways during PMA-induced MK differentiation and ploidy development. The present study demonstrates that TGF-βR-induced non-canonical ERK1/2 and PI3K/AKT pathways are associated with PMA-induced MK differentiation and ploidy development; in this molecular mechanism, JPX lncRNA could act as a decoy for miR-9-5p, miR-17-5p, and miR-106-5p, titrating them away from TGF-βR mRNAs. Importantly, this study reveals the activation of ERK1/2 and PI3K/AKT pathway in PMA-induced Dami cell differentiation into MK. The identified differentially expressed lncRNA signatures may facilitate further study of the detailed molecular mechanisms associated with MK development. Thus, our data provide numerous targets with therapeutic potential for the modulation of the differentiation of megakaryoblastic cells in AMKL.

Effects of the Latex of Synadenium grantii Hook F. (Euphorbiaceae) on a Preclinical Model of Canine Prostate Cancer.

Prostatic cancer (PC) stands out in terms of its occurrence, pathophysiology, and unfavorable prognostics in humans and dogs. Natural drugs bear an integrative potential for conventional antineoplastic treatments. In this context, the bioproducts of Synadenium grantii have been empirically used in different parts of Brazil for the integrative treatment of prostate cancer in humans. However, there is no availability of scientific evidence of the antitumor effects of S. grantii. Therefore, this study aimed to investigate the bioactive compounds in the latex of S. grantii using the high-resolution mass spectrophotometry (HRMS) and to evaluate its cytotoxic effects on primary canine PC cell cultures. Four fragments of phorbol ester were identified as potential bioactive compounds using the HRMS. With the help of an MTT ([3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide]) assay, two canine prostatic carcinoma cell lines (PC 1 and PC2) showed a decrease in the tumor cell count, with an Inhibitory concentration 50 (IC50)of 0.8469 and 0.6068 mg/ml, respectively, for PC1 and PC2. In conclusion, the latex of S. grantii contains phorbol esters in its composition, and its aqueous solution has a cytotoxic effect on canine metastatic PC cells in vitro.

Acetylcholine Receptor Stimulation Activates Protein Kinase C Mediated Internalization of the Dopamine Transporter.

The dopamine transporter (DAT) clears neurotransmitters from the extracellular space and serves as an important regulator of signal amplitude and duration at sites of dopamine release. Several different intracellular signaling pathways have been observed to modulate DAT activity through the regulation of the trafficking of the carriers to and from the cell surface. Acute activation of protein kinase C (PKC) by phorbol esters facilitates clathrin-dependent internalization of the DAT in a variety of model systems; however, the physiological stimuli and cell-surface receptor systems that activate PKC and regulate the DAT in dopamine neurons remain elusive. We report here that stimulation of M1/M5 muscarinic receptors in midbrain cultures decreases the ability of dopamine neurons to transport dopamine through DAT. Application of the cholinomimetic drug carbachol leads to a decrease in DAT activity in primary cultures while the M1/M5-specific antagonist, pirenzepine, blocks these effects. The M3 antagonist, DAU 5884, does not affect, but a positive modulator of M5, VU 0238429, enhances the loss of DAT function in response to carbachol and acetylcholine. These data implicate M1/M5 receptors on dopamine neurons in the modulation of DAT function. Bisindolylmaleimide, a PKC inhibitor, blocks the effects of carbachol stimulation on dopamine uptake, supporting a role for PKC in muscarinic receptor-mediated DAT internalization. Furthermore, as shown previously for PKC-induced internalization, downregulation of the DAT is dependent on both clathrin and dynamin. A Gq-specific inhibitor peptide also blocks the effects of carbachol on DAT in primary cultures, confirming Gq as the G-protein that couples M1/M5 receptors to PKC activation in these cells. In acute midbrain slices, biotinylation of cell-surface proteins revealed the loss of dopamine transport mediated by muscarinic receptor stimulation was, indeed, due to loss of membrane expression of the DAT in endogenous tissue. These data indicate that stimulation of cholinergic pathways can lead to modulation of dopamine through internalization of the DAT.

Probing the Diacylglycerol Binding Site of Presynaptic Munc13-1.

Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl-sn-glycerol (DOG)- and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.

AMPK Inhibits mTOR-Driven Keratinocyte Proliferation after Skin Damage and Stress

Epidermal keratinocytes (KCs) rapidly proliferate to repair the skin barrier, and a strict control of division is necessary for healthy tissue homeostasis. However, the pathways that restrain proliferation after epidermal stress are not known. AMPK is an important signaling mediator of energy metabolism previously associated with skin stress and cancer; yet, its explicit impact on KC growth is not known. To examine the requirement of epidermal AMPK in physiologic skin repair, we genetically deleted AMPK within all adult, keratin 14‒expressing KCs of mice. AMPK loss resulted in hyperproliferation and hyperactive mTOR signaling after acute wounding, UVB exposure, and phorbol ester application. This excessive division could be completely blocked by the mTORC1 inhibitor rapamycin. Moreover, we establish that the diabetes drug metformin depends on AMPK to suppress stress-induced KC proliferation. Collectively, these findings show that KC AMPK restrains mTORC1 to control epidermal proliferation after tissue injury.