Papers by Kristof Van Kolen
induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibril... more induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibrillization by Aβ, providing potent seeds for propagating Tau pathology in vivo, as demonstrated here, provides a compelling molecular mechanism for Aβ-induced propagation of Tau-pathology, beyond regions with pre-existing Tau-pathology (entorhi-nal cortex/locus coeruleus). Cross-seeding along functional connections could thereby resolve the initial spatial disso-ciation between amyloid-and Tau-pathology, and preferential propagation of Tau-pathology in regions with pre-existing 'silent' Tau-pathology, by conversion of a 'silent' Tau pathology to a 'spreading' Tau-pathology, observed in AD.
Molecular Endocrinology, Jun 1, 2008
Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to ... more Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.
J Neurochem, 2004
Cyclic AMP-dependent induction of differentiation by activation of the b-adrenergic receptor is c... more Cyclic AMP-dependent induction of differentiation by activation of the b-adrenergic receptor is correlated with inhibition of protein kinase B activity concomitant with growth arrest and increase in glial fibrillary acidic protein (GFAP) synthesis in rat C6 glioma cells. Costimulation of the b-adrenergic receptor with purinergic receptors activated by 2-methylthio-adenosine-5¢-diphosphate (2MeSADP) increased protein kinase B (PKB) phosphorylation above the level measured in nonstimulated cells and abolished cAMP-dependent differentiation. Transfection of cells with constitutively active PKB confirmed that reactivation of PKB is involved in the 2MeS-ADP-dependent inhibition of GFAP synthesis. The P2Y 12 and P2Y 13 receptor antagonist AR-C69931MX [N 6 -(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-b,c-dichloro-methylene ATP] decreased PKB phosphorylation to the level in non-stimulated cells, whereas the P2Y 13 antagonists pyridoxalphosphate-6-azophenyl-2¢,4¢-disulfonic acid (PPADS) and P 1 ,P 3di(adenosine-5¢) tetraphosphate (Ap 4 A) did not alter the 2MeSADP-induced phosphorylation of PKB, showing that enhanced PKB activity and subsequent phosphorylation of glycogen synthase kinase-3 is due to stimulation of the P2Y 12 receptor. In addition, experiments in the presence of pertussis toxin and phosphatidylinositol 3-kinase (PI 3-K) activity assays demonstrated that the P2Y 12 receptor-mediated increase in PKB phosphorylation is G i protein-and PI 3-K-dependent. The presented data demonstrated that a cAMP-dependent inhibition of PKB induces differentiation of C6 glioma cells and that inhibition of adenylate cyclase and reactivation of the PI 3-K/ PKB pathway by the P2Y 12 receptor reverses differentiation into enhanced proliferation.
Acta Neuropathologica, 2016
Genetic, clinical, histopathological and biomarker data strongly support Beta-amyloid (Aβ) induce... more Genetic, clinical, histopathological and biomarker data strongly support Beta-amyloid (Aβ) induced spreading of Tau-pathology beyond entorhinal cortex (EC), as a crucial process in conversion from preclinical cognitively normal to Alzheimer's Disease (AD), while the underlying mechanism remains unclear. In vivo preclinical models have reproducibly recapitulated Aβ-induced Tau-pathology. Tau pathology was thereby also induced by aggregated Aβ, in functionally connected brain areas, reminiscent of a prion-like seeding process. In this work we demonstrate, that pre-aggregated Aβ can directly induce Tau fibrillization by cross-seeding, in a cell-free assay, comparable to that demonstrated before for alpha-synuclein and Tau. We furthermore demonstrate, in a well-characterized cellular Tau-aggregation assay that Aβ-seeds cross-seeded Tau-pathology and strongly catalyzed pre-existing Tau-aggregation, reminiscent of the pathogenetic process in AD. Finally, we demonstrate that heterotypic seeded Tau by pre-aggregated Aβ provides efficient seeds for induction and propagation of Tau-pathology in vivo. Prion-like, heterotypic seeding of Tau fibrillization by Aβ, providing potent seeds for propagating Tau pathology in vivo, as demonstrated here, provides a compelling molecular mechanism for Aβ-induced propagation of Tau-pathology, beyond regions with pre-existing Tau-pathology (entorhinal cortex/locus coeruleus). Cross-seeding along functional connections could thereby resolve the initial spatial dissociation between amyloid- and Tau-pathology, and preferential propagation of Tau-pathology in regions with pre-existing 'silent' Tau-pathology, by conversion of a 'silent' Tau pathology to a 'spreading' Tau-pathology, observed in AD.
Alzheimer's & Dementia, 2014
Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tau... more Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a timedependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.
Journal of neurochemistry, 2004
Cyclic AMP-dependent induction of differentiation by activation of the beta-adrenergic receptor i... more Cyclic AMP-dependent induction of differentiation by activation of the beta-adrenergic receptor is correlated with inhibition of protein kinase B activity concomitant with growth arrest and increase in glial fibrillary acidic protein (GFAP) synthesis in rat C6 glioma cells. Costimulation of the beta-adrenergic receptor with purinergic receptors activated by 2-methylthio-adenosine-5'-diphosphate (2MeSADP) increased protein kinase B (PKB) phosphorylation above the level measured in non-stimulated cells and abolished cAMP-dependent differentiation. Transfection of cells with constitutively active PKB confirmed that reactivation of PKB is involved in the 2MeSADP-dependent inhibition of GFAP synthesis. The P2Y(12) and P2Y(13) receptor antagonist AR-C69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloro-methylene ATP] decreased PKB phosphorylation to the level in non-stimulated cells, whereas the P2Y(13) antagonists pyridoxalphosphate-6-azophenyl-2',4...
Neurobiology of Disease, 2015
Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tau... more Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a timedependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.
FEBS Journal, 2006
When nucleotide hydrolysis is prevented, agonists of the P2Y 12 receptor enhance the proliferatio... more When nucleotide hydrolysis is prevented, agonists of the P2Y 12 receptor enhance the proliferation of C6 glioma cells by RhoA-dependent, protein kinase C (PKC)-dependent activation of the extracellular signal-regulated kinase ERK) pathway [Claes P, Grobben B, Van Kolen K, Roymans D & Slegers H (2001) Br J Pharmacol 134, 402-408; Grobben B, Claes P, Van Kolen K, Roymans D, Fransen P, Sys SU & Slegers H (2001) J Neurochem 78, [1325][1326][1327][1328][1329][1330][1331][1332][1333][1334][1335][1336][1337][1338]. In this study, we show that ERK1 ⁄ 2 phosphorylation was not affected by transfection of the cells with the Gbc-subunit-scavenging adrenergic receptor kinase peptide [bARK1-(495-689)] or with Rap1-GAPII, indicating that P2Y 12 receptor stimulation enhances ERK1 ⁄ 2 phosphorylation by G i a subunit-mediated signaling independently of Rap1 activation. Inhibition of the RhoA downstream effector Rho-associated coiled-coil-containing kinase (ROCK) with Y-27632 did not affect the P2Y 12 receptor-induced increase in ERK1 ⁄ 2 phosphorylation but abrogated the mitogenic response. Involvement of growth factor receptor transactivation in the signaling towards ERK phosphorylation could be ruled out by the lack of an effect of PP2, AG1024, AG1296 or SU1498, inhibitors of Src, insulin-like growth factor receptor, platelet-derived growth factor receptor and vascular endothelial growth factor receptor kinase activity, respectively. Experiments with bisindolylmaleimide I and IX indicated the requirement of PKC activity. Classical and novel PKC isoforms could be excluded by treatment of the cells with Go¨6976 and calphostin C, whereas addition of a myristoylated PKCf pseudosubstrate inhibitor completely abolished P2Y 12 receptor-induced ERK1 ⁄ 2 activation. Moreover, coimmunoprecipitation experiments revealed PKCf ⁄ Raf1 and PKCf ⁄ ERK association, indicating the involvement of PKCf. From the data presented, we can conclude that the P2Y 12 receptor enhances cell proliferation by a G i a-dependent, RhoA-dependent PKCf ⁄ Raf1 ⁄ MEK ⁄ ERK pathway that requires activation of ROCK, which is not involved in ERK1 ⁄ 2 signaling.
Purinergic Signalling, 2006
The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been kno... more The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed.
Neuropharmacology, 2010
CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression... more CRF-induced ERK phosphorylation has been shown to be an important mechanism underlying expression of pro-opiomelanocortin, a key precursor molecule in the hypothalamic pituitary adrenal axis. In AtT20 cells, CRF signalling has been investigated but the mechanism behind CRF-induced ERK activity is not fully understood. This paper elucidates the signalling cascade involved in this phenomenon. Involvement of CRF(1) receptor on ERK phosphorylation was shown by using CRF and urocortin 1. The lack of inhibitory effect of pertussis toxin and BAPTA-AM excluded involvement of G(i)-coupling and calcium mobilization respectively. In contrast, the process is suggested to be driven by cAMP since treatment of AtT20 cells with forskolin triggered strong ERK phosphorylation. Treatment with PKA inhibitors had a minor effect on CRF-induced ERK signalling while phosphorylation of CREB was completely abolished. This ruled out involvement of PKA and suggested a role for exchange protein directly activated by cAMP (EPAC). Moreover, an activator of EPACs 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate mimicked CRF-induced ERK phosphorylation. Gene expression analysis showed high levels of EPAC2 mRNA and protein but low levels of EPAC1. Knockdown of EPAC2 expression by the use of specific siRNAs abolished CRF- and forskolin-induced ERK phosphorylation. The current study demonstrates a clear cAMP-dependent but PKA-independent mechanism underlying CRF-induced ERK activity that proceeds via EPAC2 signalling. Further research will provide more insight in the role of EPAC2 in CRF signalling.
Molecular Pharmacology, 2009
The molecular mechanisms governing calcium signal transduction of corticotropinreleasing factor (... more The molecular mechanisms governing calcium signal transduction of corticotropinreleasing factor (CRF) receptors CRF 1 and CRF 2(a) stably expressed in human embryonic kidney 293 (HEK293) cells were investigated. Calcium signaling strictly depended on intracellular calcium sources and this is the first study to establish a prominent contribution of the three major G-protein families to CRF receptor mediated calcium signaling. Overexpression of G αq/11 and G α16 led to leftward shifts of the agonist concentration response curves. Blockade of G αq/11 proteins by the siRNA technology partially reduced agonist-mediated calcium responses in CRF 1and CRF 2(a) -expressing HEK293 cells, thereby proving a contribution of the G q protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of G i/o proteins with pertussis toxin treatment. This effect was mediated by direct binding of G βγ subunits to phospholipase C. G i/o inhibition also elevated cAMP responses in CRF receptor overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF 1 and CRF 2(a)
Molecular Endocrinology, 2008
Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to ... more Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.
Journal of Neurochemistry, 2004
Cyclic AMP-dependent induction of differentiation by activation of the b-adrenergic receptor is c... more Cyclic AMP-dependent induction of differentiation by activation of the b-adrenergic receptor is correlated with inhibition of protein kinase B activity concomitant with growth arrest and increase in glial fibrillary acidic protein (GFAP) synthesis in rat C6 glioma cells. Costimulation of the b-adrenergic receptor with purinergic receptors activated by 2-methylthio-adenosine-5¢-diphosphate (2MeSADP) increased protein kinase B (PKB) phosphorylation above the level measured in nonstimulated cells and abolished cAMP-dependent differentiation. Transfection of cells with constitutively active PKB confirmed that reactivation of PKB is involved in the 2MeS-ADP-dependent inhibition of GFAP synthesis. The P2Y 12 and P2Y 13 receptor antagonist AR-C69931MX [N 6 -(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-b,c-dichloro-methylene ATP] decreased PKB phosphorylation to the level in non-stimulated cells, whereas the P2Y 13 antagonists pyridoxalphosphate-6-azophenyl-2¢,4¢-disulfonic acid (PPADS) and P 1 ,P 3di(adenosine-5¢) tetraphosphate (Ap 4 A) did not alter the 2MeSADP-induced phosphorylation of PKB, showing that enhanced PKB activity and subsequent phosphorylation of glycogen synthase kinase-3 is due to stimulation of the P2Y 12 receptor. In addition, experiments in the presence of pertussis toxin and phosphatidylinositol 3-kinase (PI 3-K) activity assays demonstrated that the P2Y 12 receptor-mediated increase in PKB phosphorylation is G i protein-and PI 3-K-dependent. The presented data demonstrated that a cAMP-dependent inhibition of PKB induces differentiation of C6 glioma cells and that inhibition of adenylate cyclase and reactivation of the PI 3-K/ PKB pathway by the P2Y 12 receptor reverses differentiation into enhanced proliferation.
Journal of Neurochemistry, 2007
glutamate; PKA, protein kinase C; PKC, protein kinase A; RACK, receptor for activated C kinase; T... more glutamate; PKA, protein kinase C; PKC, protein kinase A; RACK, receptor for activated C kinase; TRPV1, transient receptor potential vanilloid subtype 1; VDCC, voltage-dependent calcium channel.
Journal of Neurochemistry, 2001
We have previously shown that an ecto-NPPase modulates the ATP-and ADP-mediated P2Y AC -receptor ... more We have previously shown that an ecto-NPPase modulates the ATP-and ADP-mediated P2Y AC -receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap 3 A induced no detectable PI turnover and were identi®ed as speci®c agonists of the P2Y AC -receptor with EC 50 values of 250^37 pM and 1^0.5 mM, respectively. P2Y AC -receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2Y ACpurinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP 3 -independent Ca 21 -in¯ux was observed after P2Y AC -receptor activation. Inhibition of this in¯ux by Ca 21 -chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a speci®c MEK-inhibitor inhibited the 2MeSADP-and Ap 3 A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA Asn19 rendered C6 cells insensitive to P2Y AC -receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a signi®cant increase in the phosphorylation of raf-1 after P2Y AC -receptor activation. We may conclude that P2Y AC -purinoceptor agonists activate MAP kinase through a G i -RhoA-PKC-raf-MEK-dependent, but ras-and Ca 21 -independent cascade.
British Journal of Pharmacology, 2001
1 Extracellularly added P 1 ,P 3 -di(adenosine-5') triphosphate (Ap 3 A), P 1 ,P 4 -di(adenosine-... more 1 Extracellularly added P 1 ,P 3 -di(adenosine-5') triphosphate (Ap 3 A), P 1 ,P 4 -di(adenosine-5') tetraphosphate (Ap 4 A), ATP, ADP, AMP and adenosine are growth inhibitory for rat C6 glioma cells. Analysis of nucleotide hydrolysis and the use of nucleotidase inhibitors demonstrated that the latter inhibition is due to hydrolysis of the nucleotides to adenosine. 2 Agonists of the P2Y AC 7 -receptor enhance the growth of C6 cells if their hydrolysis to adenosine is inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). In these conditions, the potency to stimulate cell growth parallels the ranking of the receptor agonists, i.e. 2methylthioadenosine-5'-diphosphate (2MeSADP)4Ap 3 A4Ap 4 A. ATP and ADP are still hydrolysed in the presence of PPADS and have no proliferative eect on C6 cells. 3 The enhanced growth is due to a P2Y AC 7 -receptor-mediated activation of p42/44 mitogen-activated protein kinase (MAPK) as shown by immunoblotting and protein kinase assays for active MAPK and the use of the MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059. 4 The UTP-induced enhancement of the growth of C6 cells is due to activation of MAPK by a PPADS sensitive nucleotide receptor. 5 In conclusion, the eect of nucleotides on the growth of C6 cells is determined by ectonucleotidases and by activation of nucleotide receptors. Hydrolysis of nucleotides to adenosine induces growth inhibition while inhibition of the hydrolysis of agonists of the P2Y AC 7 -receptor enhances cell growth by activation of MAPK.
Biochemical Pharmacology, 2004
Cyclic AMP-dependent differentiation of rat C6 glioma cells into an astrocyte type II is characte... more Cyclic AMP-dependent differentiation of rat C6 glioma cells into an astrocyte type II is characterized by inhibition of cell growth and induction of glial fibrillary acidic protein (GFAP) synthesis. Activation of the P2Y 12 receptor with 2-methylthioadenosine-5 0 -diphosphate inhibited b-adrenergic receptor-induced differentiation. The selective P2Y 12 receptor antagonist N 6 -(2-methylthioethyl)-2-(3,3,3trifluoropropylthio)-b,g-dichloromethylene ATP abolished the receptor-mediated effect on differentiation. In contrast non-selective antagonists of P2Y receptors did not revert the inhibiting effect of the P2Y 12 receptor on differentiation. Reactive blue 2 (RB2), a potent P2Y 12 receptor antagonist, completely inhibited the synthesis of GFAP, while the P2Y receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid were less efficient. However, although P2Y receptor antagonists inhibited GFAP synthesis to a different extent they were unable to relieve the growth inhibition that accompanied induction of differentiation, whereas stimulation of the P2Y 12 receptor with 2-methylthioadenosine-5 0 -diphosphate inhibited GFAP expression and restored cell proliferation. Assay of the activity of phosphatidylinositol 3-kinase (PI 3-K), an enzyme required for GFAP expression [J. Neurochem. 76 (2001) 610], showed that RB2 inhibited this enzyme after cellular uptake, while suramin and pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid inhibited PI 3-K to a lesser extent. The intracellular concentration of RB2 increased in time and attained the IC 50 for PI 3-K inhibition (4 mM) after 40min incubation with 50 mM RB2. In conclusion, cAMP-induced differentiation in C6 cells is inhibited by activation of the P2Y 12 receptor. In addition, synthesis of GFAP is also inhibited by cellular uptake of non-selective nucleotide receptor antagonists that inhibit PI 3-K, a kinase required for the cAMP-dependent induction of differentiation. # 2004 Elsevier Inc. All rights reserved. fibrillary acidic protein; MAPK, mitogen-activated protein kinase; MEM, minimal essential medium; 2MeSADP, 2-methylthioadenosine-5 0 -diphosphate; PI 3-K, phosphatidylinositol 3-kinase; PI(3)P, phosphatidylinositol 3-phosphate; PIPES, 1,4-piperazinediethanesulfonic acid; PPADS, pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid; RB2, reactive blue 2.
Alzheimer's & Dementia, 2012
This study were respectively investigated to compare the cognitive changes of MCI-no PD patients ... more This study were respectively investigated to compare the cognitive changes of MCI-no PD patients with those of MCI-PD patients. This study found that patients with MCI-PD had relatively reduced visuospatial function compared to patients with MCI-no PD. Our study is significant because we compared the cognitive dysfunction directly of the patients with MCI-no PD and MCI-PD. There should be further large-scale, prospective studies in the future.
Neuron, 2012
LRRK2 is a kinase mutated in Parkinson's disease, but how the protein affects synaptic function r... more LRRK2 is a kinase mutated in Parkinson's disease, but how the protein affects synaptic function remains enigmatic. We identified LRRK2 as a critical regulator of EndophilinA. Using genetic and biochemical studies involving Lrrk loss-of-function mutants and Parkinson-related LRRK2 G2019S gain-of-kinase function, we show that LRRK2 affects synaptic endocytosis by phosphorylating EndoA at S75, a residue in the BAR domain. We show that LRRK2-mediated EndoA phosphorylation has profound effects on EndoA-dependent membrane tubulation and membrane association in vitro and in vivo and on synaptic vesicle endocytosis at Drosophila neuromuscular junctions in vivo. Our work uncovers a regulatory mechanism that indicates that reduced LRRK2 kinase activity facilitates EndoA membrane association, while increased kinase activity inhibits membrane association. Consequently, both too much and too little LRRK2-dependent EndoA phosphorylation impedes synaptic endocytosis, and we propose a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses.
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Papers by Kristof Van Kolen