Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
J Alzheimers Dis ; 88(1): 207-228, 2022.
Article in English | MEDLINE | ID: mdl-35570492

ABSTRACT

BACKGROUND: Deposits of hyperphosphorylated tau fibrils are hallmarks of a broad spectrum of tauopathies, including Alzheimer's disease (AD). OBJECTIVE: To investigate heterogeneity of tau pathology across brain extracts from a broad selection of different tauopathies and examine the binding properties of the humanized pS396-tau antibody hC10.2 and six other anti-tau antibodies. METHODS: 76 individual tauopathy tissue samples were analyzed in a battery of assays: immunohistochemistry, ELISA, tau aggregation assay, western blot, [3H]PI-2620 and [3H]MK-6240 tau tracer binding, and aggregated seeding activity in RD_P301S HEK293T Biosensor cells. The efficiency of seven anti-tau antibodies to engage with pathological tau species was directly compared. RESULTS: Our data indicate that a strong correlation existed between the tau tracer binding, amount of tau aggregates, pS396-tau phosphorylation, and seeding activity. The hC10.2 antibody, which has entered clinical development, effectively engaged with its epitope across all individual cases of mid-stage and late AD, and primary tauopathies. hC10.2 was superior compared to other phospho- and total tau antibodies to prevent seeded tau aggregation in the biosensor cells. hC10.2 effectively depleted hyperphosphorylated and aggregated tau species across all tauopathy samples proportionally to the amount of tau aggregates. In AD samples, hC10.2 bound to ghost tangles which represent extracellular pathological tau species. CONCLUSION: S396 hyperphosphorylation is a feature of the formation of seeding-competent tau across different tauopathies and it is present both in intra- and extracellular pathological tau. hC10.2 represents an excellent candidate for a hyperphosphorylation-selective therapeutic tau antibody for the treatment of AD and primary tauopathies.


Subject(s)
Alzheimer Disease , Tauopathies , Alzheimer Disease/pathology , Antibodies/metabolism , Brain/pathology , HEK293 Cells , Humans , Tauopathies/pathology , tau Proteins/metabolism
2.
Alzheimers Dement (N Y) ; 4: 521-534, 2018.
Article in English | MEDLINE | ID: mdl-30386817

ABSTRACT

INTRODUCTION: The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. METHODS: Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. RESULTS: The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. DISCUSSION: Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

3.
Sci Rep ; 8(1): 6209, 2018 04 18.
Article in English | MEDLINE | ID: mdl-29670132

ABSTRACT

Tau antibodies have shown therapeutic potential for Alzheimer's disease and several are in clinical trials. As a microtubule-associated protein, tau relies on dynamic phosphorylation for its normal functions. In tauopathies, it becomes hyperphosphorylated and aggregates into toxic assemblies, which collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser396 has received particular attention because of its prominence and stability in tauopathies. Here we report the first structure of a monoclonal tau antibody in complex with the pathologically important phospho-Ser396 residue. Its binding region reveals tau residues Tyr394 to phospho-Ser396 stabilized in a ß-strand conformation that is coordinated by a phospho-specific antigen binding site. These details highlight a molecular switch that defines this prominent conformation of tau and ways to target it. Overall, the structure of the antibody-antigen complex clarifies why certain phosphorylation sites in tau are more closely linked to neurodegeneration than others.


Subject(s)
Antibodies, Monoclonal/chemistry , Models, Molecular , Protein Conformation , tau Proteins/chemistry , Alzheimer Disease , Amino Acid Motifs , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibody Specificity/immunology , Binding Sites , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Peptides/chemistry , Peptides/immunology , Phosphorylation , Protein Binding , Structure-Activity Relationship , tau Proteins/immunology , tau Proteins/metabolism
4.
Brain Res ; 1565: 63-73, 2014 May 27.
Article in English | MEDLINE | ID: mdl-24675027

ABSTRACT

Tissue-type plasminogen activator (t-PA) is the only thrombolytic treatment available for patients with acute ischaemic stroke. However, t-PA can increase permeability of the blood-brain barrier (BBB). Desmoteplase is a plasminogen activator derived from the common vampire bat, currently under clinical development for ischaemic stroke. We compared how t-PA and desmoteplase influenced BBB permeability using a human in vitro model where primary brain endothelial cells (BEC) and astrocytes are co-cultured on the opposite sides of a porous membrane. Permeability changes were evaluated 6 or 24h post-stimulation by passage of fluorescent albumin across the membrane. Under normoxic conditions, t-PA, but not desmoteplase, increased BBB permeability. Surprisingly, the ability of t-PA to affect the barrier was lost under conditions of oxygen-glucose deprivation (OGD). Addition of plasminogen re-sensitised the BBB to the action of t-PA under both normoxia and OGD, but did not affect the inert behaviour of desmoteplase, even when digested fibrinogen was added to ensure optimal plasmin generation. These observations coincided with plasmin-dependent changes in astrocyte and BEC morphology and disruption of tight junction proteins in BECs, specifically initiated by t-PA but not by desmoteplase. Finally, inhibition of plasmin post-stimulation with t-PA and plasminogen, especially within 2h, protected the BBB against t-PA-mediated barrier opening. Hence t-PA, but not desmoteplase, increases BBB permeability under both normoxic and OGD conditions in a reversible, plasmin-dependent process. The inability of desmoteplase to increase permeability despite its capacity to generate plasmin provides further support for its use as thrombolytic in patients with ischaemic stroke.


Subject(s)
Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Fibrinolytic Agents/pharmacology , Plasminogen Activators/pharmacology , Plasminogen/pharmacology , Tissue Plasminogen Activator/pharmacology , Brain/drug effects , Brain/metabolism , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Glucose/metabolism , Humans , Oxygen/metabolism , Permeability
5.
Mol Pharmacol ; 82(5): 929-37, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22899869

ABSTRACT

Metabotropic glutamate receptor subtype 5 (mGluR5) is a potential drug target in neurological and psychiatric disorders, and subtype-selective allosteric modulators have attracted much attention as potential drug candidates. In this study, the binding sites of three novel 2-methyl-6-(phenylethynyl)pyridine (MPEP)-derived negative allosteric modulators, 2-, 3-, and 4-BisPEB, have been characterized. 2-, 3-, and 4-BisPEB are 1,3-bis(pyridinylethynyl)-benzenes and differ only by the position of the nitrogen atoms in the pyridine rings. Despite their high structural similarity, 2-BisPEB [1,3-bis(pyridin-2-ylethynyl)-benzene, nitrogen atoms in ortho positions], with an IC(50) value in the nanomolar range, is significantly more potent than the 3- and 4-pyridyl analogs. Mutational analysis, directed by a previously published mGluR5 homology model, was used to determine key residues for the ligand-receptor interactions that may explain the potency differences of 2-, 3-, and 4-BisPEB. Residues Ile651, Pro655, Tyr659, Asn747, Trp785, Phe788, Tyr792, Ser809, and Ala810 were found to have critical roles for the activity of one or more of the three BisPEBs and the reference compound MPEP. The mutagenesis data suggest that the higher potency of 2-BisPEB is due to hydrogen bonding to Ser809 because the S809A mutation made 2-BisPEB equipotent to 3- and 4-BisPEB (IC(50), 1-2.5 µM). The potency of MPEP was also greatly affected by S809A (52-fold), suggesting that a Ser809-mediated hydrogen bond is also a key interaction between MPEP and mGluR5. Potential binding modes of 2-, 3-, and 4-BisPEB obtained by molecular docking to the mGluR5 homology model provide a structural context for the reported major mutational effects.


Subject(s)
Alkynes/chemical synthesis , Benzene Derivatives/chemistry , Cholinergic Agents/chemistry , Molecular Docking Simulation , Pyridines/chemistry , Receptors, Metabotropic Glutamate/metabolism , Alkynes/pharmacology , Allosteric Regulation , Animals , Benzene Derivatives/pharmacology , Binding Sites , Calcium/metabolism , Cell Line , Cholinergic Agents/pharmacology , Cricetinae , Humans , Hydrogen Bonding , Point Mutation , Pyridines/pharmacology , Radioligand Assay , Receptor, Metabotropic Glutamate 5 , Receptors, Metabotropic Glutamate/genetics , Structure-Activity Relationship
6.
Biochim Biophys Acta ; 1774(6): 701-13, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17499031

ABSTRACT

Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composition. A large set of available synthetic peptides (n=127) was tested for binding to calreticulin and the results analysed by multivariate data analysis. The parameter that correlated best with binding was hydrophobicity while beta-turn potential disfavoured binding. Only hydrophobic peptides longer than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has a peptide-binding specificity for hydrophobic sequences and delineate the fine specificity of calreticulin for hydrophobic amino acid residues.


Subject(s)
Calreticulin/metabolism , Molecular Chaperones/metabolism , Peptide Fragments/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/metabolism , Histocompatibility Antigens Class I/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Peptide Fragments/chemistry , Prions/chemistry , Prions/metabolism , Protein Binding , Sensitivity and Specificity , Serum Amyloid P-Component/chemistry , Serum Amyloid P-Component/metabolism
7.
Science ; 305(5681): 239-42, 2004 Jul 09.
Article in English | MEDLINE | ID: mdl-15247477

ABSTRACT

Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype-selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.


Subject(s)
Erythropoietin/analogs & derivatives , Erythropoietin/therapeutic use , Nervous System Diseases/drug therapy , Neuroprotective Agents/therapeutic use , Animals , Apoptosis , Binding Sites , Cells, Cultured , Diabetic Neuropathies/drug therapy , Drug Design , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Erythropoiesis , Erythropoietin/chemistry , Erythropoietin/genetics , Erythropoietin/metabolism , Erythropoietin/pharmacology , Female , Hematocrit , Humans , Ligands , Mice , Mice, Inbred C3H , Mutagenesis , Neurons/metabolism , Neuroprotective Agents/chemistry , Neuroprotective Agents/metabolism , Neuroprotective Agents/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Erythropoietin/metabolism , Recombinant Proteins , Signal Transduction , Spinal Cord Compression/drug therapy , Stroke/drug therapy , Structure-Activity Relationship
8.
Semin Cancer Biol ; 13(6): 449-59, 2003 Dec.
Article in English | MEDLINE | ID: mdl-15001164

ABSTRACT

Interleukin 2 (IL-2) is a promising immunotherapeutic agent for the treatment of metastatic melanoma and renal cell carcinoma. Systemic administration of high dose IL-2 produces objective responses in up to 25% of melanoma patients, and a low but significant proportion of these patients experience durable responses. Nevertheless, the cells and molecules responsible for induction of tumor regression over the course of IL-2 treatment remain unknown. New strategies in tumor immunotherapy have evolved over the past decade as a consequence of significant progress in the field, in particular with respect to the characterization of peptide epitopes derived from tumor associated antigens, and the role of antigen presenting cells in the initiation of cellular immune responses. Alongside with these factual as well as conceptual advances, new methods have been developed to monitor and characterize anti-tumor T cell responses in cancer patients. Application of these tools to dissect anti-tumor responses has demonstrated that various immune therapeutic approaches can induce powerful systemic anti-tumor cytotoxic T lymphocyte (CTL) responses. However, only limited efforts have been made to use present days tool to analyze anti-tumor immune responses in patients treated with IL-2 based immunotherapy. We have examined CTL responses against known tumor antigens in melanoma patients over the course of IL-2 based immunotherapy (electrochemotherapy). Surprisingly, anti-tumor CTL responses significantly declined upon initiation of therapy, but reappeared when IL-2 administration was paused. Molecular analyses of the clonotypic composition of responding T cells demonstrated that new clones emerged over the course of treatment, and that tumor-specific T cells that had left the peripheral blood could subsequently be detected at the tumor site. These data provide new insight into the biological actions of IL-2 and highlight the difficulties associated with the monitoring of anti-tumor immune responses. This underlines the importance of frequent sampling of blood and tumor biopsies to be analyzed with a combination of state of the art technologies in order to gain detailed information on the interactions between cancer cells and cells of the immune system.


Subject(s)
Antineoplastic Agents/immunology , Interleukin-2/immunology , Melanoma/immunology , Melanoma/therapy , Skin Neoplasms/immunology , Skin Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , Antigens, Neoplasm/immunology , Humans , Immunotherapy/methods , T-Lymphocytes, Cytotoxic/classification , Treatment Outcome
SELECTION OF CITATIONS
SEARCH DETAIL
...