The Experts below are selected from a list of 738 Experts worldwide ranked by ideXlab platform
Bernardo Rudy - One of the best experts on this subject based on the ideXlab platform.
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DPP6 regulation of dendritic morphogenesis impacts hippocampal synaptic development
Nature Communications, 2013Co-Authors: Ben Throesch, Bernardo Rudy, Faith Kung, Jameice T Decoster, Cory J Berner, Richard E Cheney, Dax A HoffmanAbstract:Dipeptidyl-peptidase 6 is implicated in different neuropsychiatric pathologies. Lin and colleagues genetically delete dipeptidyl-peptidase 6 in mice and find that this results in impaired development of dendritic filopodia, as well as a reduction in the dendritic tree size, spine density and functional synapses.
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DPP6 establishes the a type k current gradient critical for the regulation of dendritic excitability in ca1 hippocampal neurons
Neuron, 2011Co-Authors: Jon K Maffie, Bernardo Rudy, Ronald S Petralia, Dax A HoffmanAbstract:Summary Subthreshold-activating A-type K + currents are essential for the proper functioning of the brain, where they act to delay excitation and regulate firing frequency. In CA1 hippocampal pyramidal neuron dendrites, the density of A-type K + current increases with distance from the soma, playing an important role in synaptic integration and plasticity. The mechanism underlying this gradient has, however, remained elusive. Here, dendritic recordings from mice lacking the Kv4 transmembrane auxiliary subunit DPP6 revealed that this protein is critical for generating the A-current gradient. Loss of DPP6 led to a decrease in A-type current, specifically in distal dendrites. Decreased current density was accompanied by a depolarizing shift in the voltage dependence of channel activation. Together these changes resulted in hyperexcitable dendrites with enhanced dendritic AP back-propagation, calcium electrogenesis, and induction of synaptic long-term potentiation. Despite enhanced dendritic excitability, firing behavior evoked by somatic current injection was mainly unaffected in DPP6-KO recordings, indicating compartmentalized regulation of neuronal excitability.
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the dipeptidyl peptidase like protein DPP6 determines the unitary conductance of neuronal kv4 2 channels
The Journal of Neuroscience, 2009Co-Authors: Yuri A Kaulin, Marcela S Nadal, Bernardo Rudy, Jose A De Santiagocastillo, Carmen A Rocha, Manuel CovarrubiasAbstract:The neuronal subthreshold-operating A-type K+ current regulates electrical excitability, spike timing, and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (γ) of neuronal somatodendritic A-type K+ channels composed of Kv4 pore-forming subunits is larger (∼7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (∼4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like protein-6 DPP6-S to the γ of native [cerebellar granule neuron (CGN)] and reconstituted Kv4.2 channels. Coexpression of Kv4.2 proteins with DPP6-S was sufficient to match the γ of native CGN channels; and CGN Kv4 channels from DPP6 knock-out mice yielded a γ indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in γ. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the DPP6 gene.
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the dipeptidyl peptidase like protein DPP6 determines the unitary conductance of neuronal kv4 2 channels
Biophysical Journal, 2009Co-Authors: Yuri A Kaulin, Marcela S Nadal, Bernardo Rudy, Jose A De Santiagocastillo, Carmen A Rocha, Manuel CovarrubiasAbstract:The neuronal subthreshold-operating A-type K+ current regulates electrical excitability, spike timing and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (γ) of neuronal somatodendritic A-type K+ channels composed of Kv4 pore-forming subunits is larger (∼7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (∼4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like-protein-6 DPP6-S to the γ of native (cerebellar granule neuron, CGN) and reconstituted Kv4.2 channels. Co-expression of Kv4.2 proteins with DPP6-S was sufficient to match the γ of native CGN channels; and CGN Kv4 channels from DPP6 knock-out mice yielded a γ indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in γ. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the DPP6 gene.This work was supported by grants from the National Institutes of Health (R01 NS032337-13 to MC; and NS045217 and NS30989 to BR).
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a novel DPP6 isoform DPP6 e can account for differences between neuronal and reconstituted a type k channels
Neuroscience Letters, 2009Co-Authors: Jonathon Maffie, Timothy A Blenkinsop, Bernardo RudyAbstract:The channels mediating most of the somatodendritic A-type K+ current in neurons are thought to be ternary complexes of Kv4 pore-forming subunits and two types of auxiliary subunits, the K+ channel interacting proteins (KChIPs) and dipeptidyl-peptidase-like (DPPL) proteins. The channels expressed in heterologous expression systems by mixtures of Kv4.2, KChIP1 and DPP6-S resemble in many properties the A-type current in hippocampal CA1 pyramidal neurons and cerebellar granule cells, neurons with prominent A-type K+ currents. However, the native currents have faster kinetics. Moreover, the A-type currents in neurons in intermediary layers of the superior colliculus have even faster inactivating rates. We have characterized a new DPP6 spliced isoform, DPP6-E, that produces in heterologous cells ternary Kv4 channels with very fast kinetics. DPP6-E is selectively expressed in a few neuronal populations in brain including cerebellar granule neurons, hippocampal pyramidal cells and neurons in intermediary layers of the superior colliculus. The effects of DPP6-E explain past discrepancies between reconstituted and native Kv4 channels in some neurons, and contributes to the diversity of A-type K+ currents in neurons.
Dax A Hoffman - One of the best experts on this subject based on the ideXlab platform.
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DPP6 loss impacts hippocampal synaptic development and induces behavioral impairments in recognition learning and memory
Frontiers in Cellular Neuroscience, 2018Co-Authors: Jonathan G Murphy, Rosemarie Karlsson, Ronald S Petralia, Jakob J Gutzmann, Daniel Abebe, Yaxian Wang, Heather A Cameron, Dax A HoffmanAbstract:DPP6 is well known as an auxiliary subunit of Kv4-containing, A-type K+ channels which regulate dendritic excitability in hippocampal CA1 pyramidal neurons. We have recently reported, however, a novel role for DPP6 in regulating dendritic filopodia formation and stability, affecting synaptic development and function. These results are notable considering recent clinical findings associating DPP6 with neurodevelopmental and intellectual disorders. Here we assessed the behavioral consequences of DPP6 loss. We found that DPP6 knockout (DPP6-KO) mice are impaired in hippocampus-dependent learning and memory. Results from the Morris water maze and T-maze tasks showed that DPP6-KO mice exhibit slower learning and reduced memory performance. DPP6 mouse brain weight is reduced throughout development compared with WT, and in vitro imaging results indicated that DPP6 loss affects synaptic structure and motility. Taken together, these results show impaired synaptic development along with spatial learning and memory deficiencies in DPP6-KO mice.
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DPP6 domains responsible for its localization and function
Journal of Biological Chemistry, 2014Co-Authors: Laura K Long, Michael M Hatch, Dax A HoffmanAbstract:Abstract Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K+ channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER.
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DPP6 regulation of dendritic morphogenesis impacts hippocampal synaptic development
Nature Communications, 2013Co-Authors: Ben Throesch, Bernardo Rudy, Faith Kung, Jameice T Decoster, Cory J Berner, Richard E Cheney, Dax A HoffmanAbstract:Dipeptidyl-peptidase 6 is implicated in different neuropsychiatric pathologies. Lin and colleagues genetically delete dipeptidyl-peptidase 6 in mice and find that this results in impaired development of dendritic filopodia, as well as a reduction in the dendritic tree size, spine density and functional synapses.
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DPP6 establishes the a type k current gradient critical for the regulation of dendritic excitability in ca1 hippocampal neurons
Neuron, 2011Co-Authors: Jon K Maffie, Bernardo Rudy, Ronald S Petralia, Dax A HoffmanAbstract:Summary Subthreshold-activating A-type K + currents are essential for the proper functioning of the brain, where they act to delay excitation and regulate firing frequency. In CA1 hippocampal pyramidal neuron dendrites, the density of A-type K + current increases with distance from the soma, playing an important role in synaptic integration and plasticity. The mechanism underlying this gradient has, however, remained elusive. Here, dendritic recordings from mice lacking the Kv4 transmembrane auxiliary subunit DPP6 revealed that this protein is critical for generating the A-current gradient. Loss of DPP6 led to a decrease in A-type current, specifically in distal dendrites. Decreased current density was accompanied by a depolarizing shift in the voltage dependence of channel activation. Together these changes resulted in hyperexcitable dendrites with enhanced dendritic AP back-propagation, calcium electrogenesis, and induction of synaptic long-term potentiation. Despite enhanced dendritic excitability, firing behavior evoked by somatic current injection was mainly unaffected in DPP6-KO recordings, indicating compartmentalized regulation of neuronal excitability.
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kv4 accessory protein dppx DPP6 is a critical regulator of membrane excitability in hippocampal ca1 pyramidal neurons
Journal of Neurophysiology, 2008Co-Authors: Marcela S Nadal, Bernardo Rudy, Ann M Clemens, Matthew Baron, Sungcherl Jung, Yoshio Misumi, Dax A HoffmanAbstract:A-type K+ currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic integration, action potential (AP) shape and firing patterns. In hippocampal CA1 pyramidal neurons, Kv4 channels make up the majority of the somatodendritic A-type current. Studies in heterologous expression systems have shown that Kv4 channels interact with transmembrane dipeptidyl-peptidase-like proteins (DPPLs) to regulate the surface trafficking and biophysical properties of Kv4 channels. To investigate the influence of DPPLs in a native system, we conducted voltage-clamp experiments in patches from CA1 pyramidal neurons expressing short-interfering RNA (siRNA) targeting the DPPL variant known to be expressed in hippocampal pyramidal neurons, DPPX (siDPPX). In accordance with heterologous studies, we found that DPPX downregulation in neurons resulted in depolarizing shifts of the steady-state inactivation and activation curves, a shallower conductance-voltage slope, slowed inactivation, and a delayed recovery from inactivation for A-type currents. We carried out current-clamp experiments to determine the physiological effect of the A-type current modifications by DPPX. Neurons expressing siDPPX exhibited a surprisingly large reduction in subthreshold excitability as measured by a decrease in input resistance, delayed time to AP onset, and an increased AP threshold. Suprathreshold DPPX downregulation resulted in slower AP rise and weaker repolarization. Computer simulations supported our experimental results and demonstrated how DPPX remodeling of A-channel properties can result in opposing sub- and suprathreshold effects on excitability. The Kv4 auxiliary subunit DPPX thus acts to increase neuronal responsiveness and enhance signal precision by advancing AP initiation and accelerating both the rise and repolarization of APs.
Manuel Covarrubias - One of the best experts on this subject based on the ideXlab platform.
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A novel N-terminal motif of dipeptidyl peptidase-like proteins produces rapid inactivation of KV4.2 channels by a pore-blocking mechanism.
Channels, 2020Co-Authors: Henry H. Jerng, Manuel Covarrubias, Kevin Dougherty, Paul J. PfaffingerAbstract:The somatodendritic subthreshold A-type K+ current in neurons (ISA) depends on its kinetic and voltage-dependent properties to regulate membrane excitability, action potential repetitive firing, and signal integration. Key functional properties of the Kv4 channel complex underlying ISA are determined by dipeptidyl peptidase-like proteins known as dipeptidyl peptidase 6 (DPP6) and dipeptidyl peptidase 10 (DPP10). Among the multiple known DPP10 isoforms with alternative N-terminal sequences, DPP10a confers exceptionally fast inactivation to Kv4.2 channels. To elucidate the molecular basis of this fast inactivation, we investigated the structure-function relationship of the DPP10a N-terminal region and its interaction with the Kv4.2 channel. Here, we show that DPP10a shares a conserved N-terminal sequence (MNQTA) with DPP6a (aka DPP6-E), which also induces fast inactivation. Deletion of the NQTA sequence in DPP10a eliminates this dramatic fast inactivation, and perfusion of MNQTA peptide to the cytoplasmic f...
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Dipeptidyl-peptidase-like proteins cast in a new role: enabling scorpion toxin block of A-type K+ channels
The Journal of Physiology, 2013Co-Authors: Manuel CovarrubiasAbstract:Subthreshold-operating A-type K+ currents exhibiting rapid inactivation are major regulators of somatodendritc excitability and plasticity in the nervous system (Jerng et al. 2004; Maffie & Rudy, 2008). There is also compelling evidence indicating that the Kv4.2 and Kv4.3 pore-forming subunits are significant molecular correlates of these currents (Maffie & Rudy, 2008). Although these subunits can form functional homo- and heterotetrameric A-type K+ channels, they need essential partners to recapitulate their native phenotype and perform their functions (Maffie & Rudy, 2008). Two distinct ancillary subunits are confirmed components of the Kv4 channel complex in neurones, namely the K+ channel-interacting proteins (KChIPs) and dipeptidyl-peptidase-like proteins (DPPs) encoded by various genes (KChIP1–4, DPP6 and DPP10), which generate diverse splice variants. Current structural models of the ternary Kv4 channel complex suggest a dodecameric structure, in which there is one KChIP and one DPP for every Kv4.2/Kv4.3 subunit in the pore-forming tetrameric channel (Covarrubias et al. 2008). The DPPs, in particular, affect almost every aspect in the life of the Kv4 channel complex. They increase surface expression and unitary conductance, and promote activation and inactivation gating. Dipeptidyl-peptidase-like proteins are single-pass membrane proteins with an intracellular N-terminal region and a large extracellular C-terminal region constituting ∼90% of the mass of the protein. While the N-terminal and membrane-spanning regions are mainly responsible for the gating effects, the roles of the C-terminal region are largely unknown. Recent work investigated CA1 hippocampal neurons from wild-type and DPP6 knockout (DPP6 KO) mice to determine that DPP6 establishes the somatodendritic gradient of the A-type K+ current and thereby helps regulate backpropagating action potentials and long-term potentiation (Sun et al. 2011). Whether or not the DPP6 C-terminal region influences subcellular localization is unknown. Also, despite the wealth of knowledge about the biophysical, cellular and physiological roles of DPPs, not much is known about their possible influence on the pharmacology of the ternary Kv4 channel complex. Given the extracellular topology of DPPs and their putative direct interactions with pore-forming subunits, such a role is likely. In order to investigate the functions of somatodendritic A-type K+ channels in short-term conditions, specific high-affinity blockers of neuronal Kv4 ternary complexes would be useful for both in vivo and in vitro studies. Knockdown and knockout approaches are often confounded by long-term compensation and remodelling of native conductances. In this issue of the The Journal of Physiology, Maffie et al. (2013) report compelling evidence for the new role of DPPs as determinants of the potent block of neuronal A-type K+ channels by the peptide toxin AmmTx3, a member of the α-KTX15 family of scorpion toxins. At the same time, these authors solved the apparent discrepancy between the scorpion toxin sensitivity of native and heterologously expressed Kv4.2/Kv4.3 channels, which had hindered the experimental applications of the aforementioned toxins. While the native neuronal channels are blocked by submicromolar concentrations of AmmTx3, the recombinant counterparts are only modestly inhibited by similar concentrations. At the centre of this study is the investigation of the granule neuron. A-type K+ current in cerebellar slices from wild-type and DPP6 KO mice. Clearly, 500 nm AmmTx3 nearly eliminates the A-type component of the total current. In contrast, demonstrating that high AmmTx3 sensitivity depends on the expression of DPP6, almost 90% of the A-type K+ current from the DPP6 KO mouse is resistant to the same concentration of the toxin. The authors also show that AmmTx3 is not a gating modifier and that it is most likely to act as a high-affinity external pore blocker (IC50≍ 100 nm), resembling analogous scorpion toxins acting on related Ca2+- and voltage-gated K+ channels. This important conclusion will facilitate the interpretation of results in future applications. Then, in order to show that DPP6 is definitely the culprit behind the enhanced AmmTx3 sensitivity of the neuronal A-type K+ current, the authors also reconstituted low- and high-affinity block in heterologously expressed Kv4.2 channels. Essentially, only when DPP6 was present in binary (Kv4.2+DPP6 vs. Kv4.2+KChIP1) and ternary complexes (Kv4.2+DPP6+KChIP1) did the expressed currents exhibit AmmTx3 block comparable to that observed in cerebellar granule neurons. Moreover, heterologous coexpression of Kv4.3 and DPP10 also induced A-type K+ currents exhibiting high scorpion toxin sensitivity, indicating that all known DPPs that form Kv4 channel complexes are capable of enhancing the apparent affinity of AmmTx3. How do DPPs accomplish this surprising feat? In the light of the available structural information (Covarrubias et al., 2008; Maffie & Rudy, 2008), one could imagine a role for the intriguing C-terminal region of DPPs. Like large balloons tethered to the membrane, the massive DPP dimer of dimers might hover immediately above the external vestibule of the pore but, rather than hindering toxin binding, they enhance it. Perhaps, the membrane-facing side of the extracellular C-terminal region and juxtamembrane linkers sculpt the external vestibule of the pore in Kv4.2/Kv4.3 channels to promote stronger toxin binding. Direct structural approaches will be necessary to solve this puzzling question conclusively. Additionally, it would be important to determine whether AmmTx3 also blocks native and recombinant Kv4.1 channels in a DPP-dependent manner. The Kv4.1 and Kv4.3 subunits are expressed in sensory neurons, but the subunit composition of the Kv4 channel complex in this cell type remains unknown (Phuket & Covarrubias, 2009). At any rate, the emergence of new tantalizing questions with broad implications signifies progress. Demonstrating specific DPP-dependent block of somatodendritic A-type K+ channels by AmmTx3 will undoubtedly stimulate further research on the functions, structure and pharmacology of neuronal Kv4 channel complexes and their possible roles in disease states, such as epilepsy, chronic pain, neurodegenerative disorders and autism.
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the dipeptidyl peptidase like protein DPP6 determines the unitary conductance of neuronal kv4 2 channels
The Journal of Neuroscience, 2009Co-Authors: Yuri A Kaulin, Marcela S Nadal, Bernardo Rudy, Jose A De Santiagocastillo, Carmen A Rocha, Manuel CovarrubiasAbstract:The neuronal subthreshold-operating A-type K+ current regulates electrical excitability, spike timing, and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (γ) of neuronal somatodendritic A-type K+ channels composed of Kv4 pore-forming subunits is larger (∼7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (∼4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like protein-6 DPP6-S to the γ of native [cerebellar granule neuron (CGN)] and reconstituted Kv4.2 channels. Coexpression of Kv4.2 proteins with DPP6-S was sufficient to match the γ of native CGN channels; and CGN Kv4 channels from DPP6 knock-out mice yielded a γ indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in γ. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the DPP6 gene.
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The dipeptidyl-aminopeptidase-like protein 6 is an integral voltage sensor-interacting β-subunit of neuronal KV4.2 channels
Channels, 2009Co-Authors: Kevin Dougherty, Liwei Tu, Carol Deutsch, Manuel CovarrubiasAbstract:Auxiliary β-subunits dictate the physiological properties of voltage-gated K+ (KV) channels in excitable tissues. In many instances, however, the underlying mechanisms of action are poorly understood. The dipeptidyl-aminopeptidase-like protein 6 (DPP6) is a specific β-subunit of neuronal KV4 channels, which may promote gating through interactions between the single transmembrane segment of DPP6 and the channel’s voltage sensing domain (VSD). A combination of gating current measurements and protein biochemistry (in-vitro translation and co-immunoprecipitations) revealed preferential physical interaction between the isolated KV4.2-VSD and DPP6. Significantly weaker interactions were detected between DPP6 and KV1.3 channels or the KV4.2 pore domain. More efficient gating charge movement resulting from a direct interaction between DPP6 and the KV4.2-VSD is unique among the known actions of KV channel β-subunits. This study shows that the modular VSD of a KV channel can be directly regulated by transmemb...
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the dipeptidyl peptidase like protein DPP6 determines the unitary conductance of neuronal kv4 2 channels
Biophysical Journal, 2009Co-Authors: Yuri A Kaulin, Marcela S Nadal, Bernardo Rudy, Jose A De Santiagocastillo, Carmen A Rocha, Manuel CovarrubiasAbstract:The neuronal subthreshold-operating A-type K+ current regulates electrical excitability, spike timing and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (γ) of neuronal somatodendritic A-type K+ channels composed of Kv4 pore-forming subunits is larger (∼7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (∼4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like-protein-6 DPP6-S to the γ of native (cerebellar granule neuron, CGN) and reconstituted Kv4.2 channels. Co-expression of Kv4.2 proteins with DPP6-S was sufficient to match the γ of native CGN channels; and CGN Kv4 channels from DPP6 knock-out mice yielded a γ indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in γ. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the DPP6 gene.This work was supported by grants from the National Institutes of Health (R01 NS032337-13 to MC; and NS045217 and NS30989 to BR).
Stephane Demine - One of the best experts on this subject based on the ideXlab platform.
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A nanobody-based nuclear imaging tracer targeting dipeptidyl peptidase 6 to determine the mass of human beta cell grafts in mice
Diabetologia, 2019Co-Authors: Stephane Demine, Julien Thevenet, Lorella Marselli, Piero Marchetti, François Pattou, Julie Kerr-conte, Nick Devoogdt, Rita Garcia Ribeiro, Decio L. EizirikAbstract:Aims/hypothesis Type 1 diabetes is characterised by a progressive decline in beta cell mass. This is also observed following implantation of pancreatic islet allografts, but there is no reliable information regarding the time course of beta cell loss. This is due to the limited availability of non-invasive pancreatic islet imaging techniques. We have previously described that dipeptidyl peptidase 6 (DPP6) is an alpha and beta cell-specific biomarker, and developed a camelid antibody (nanobody ‘4hD29’) against it. We demonstrated the possibility to detect DPP6-expressing cells by single-photon emission computed tomography (SPECT)/ computed tomography (CT), but the correlation between the number of cells grafted and the SPECT signal was not assessed. Here, we investigate whether the 4hD29 nanobody allows us to detect different amounts of human pancreatic islets implanted into immune-deficient mice. In addition, we also describe the adaptation of the probe for use with positron emission tomography ( PET). Methods DPP6 expression was assessed in human samples using tissue arrays and immunohistochemistry. The effect of the 4hD29 nanobody on cell death and glucose-stimulated insulin secretion was measured in EndoC-βH1 cells and in human islets using Hoechst/propidium iodide staining and an anti-insulin ELISA, respectively. We performed in vivo SPECT imaging on severe combined immunodeficient (SCID) mice transplanted with different amounts of EndoC-βH1 cells (2 × 10^6, 5 × 10^6 and 10 × 10^6 cells), human islets (1000 and 3000) or pancreatic exocrine tissue using ^99mTc-labelled 4hD29 nanobody. This DPP6 nanobody was also conjugated to N -chlorosuccinimide (NCS)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), radiolabelled with either ^67Ga (SPECT) or ^68Ga (PET) and used in a proof-of-principle experiment to detect DPP6-expressing cells (Kelly neuroblastoma) grafted in SCID mice. Results The DPP6 protein is mainly expressed in pancreatic islets. Importantly, the anti-DPP6 nanobody 4hD29 allows non-invasive detection of high amounts of EndoC-βH1 cells or human islets grafted in immunodeficient mice. This suggests that the probe must be further improved to detect lower numbers of islet cells. The 4hD29 nanobody neither affected beta cell viability nor altered insulin secretion in EndoC-βH1 cells and human islets. The conversion of 4hD29 nanobody into a PET probe was successful and did not alter its specificity. Conclusions/interpretation These findings suggest that the anti-DPP6 4hD29 nanobody may become a useful tool for the quantification of human islet grafts in mice and, pending future development, islet mass in individuals with diabetes.
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A nanobody-based nuclear imaging tracer targeting dipeptidyl peptidase 6 to determine the mass of human beta cell grafts in mice.
Diabetologia, 2019Co-Authors: Stephane Demine, Rita Garcia Ribeiro, Julien Thevenet, Lorella Marselli, Piero Marchetti, François Pattou, Julie Kerr-conte, Nick Devoogdt, Decio L. EizirikAbstract:Type 1 diabetes is characterised by a progressive decline in beta cell mass. This is also observed following implantation of pancreatic islet allografts, but there is no reliable information regarding the time course of beta cell loss. This is due to the limited availability of non-invasive pancreatic islet imaging techniques. We have previously described that dipeptidyl peptidase 6 (DPP6) is an alpha and beta cell-specific biomarker, and developed a camelid antibody (nanobody ‘4hD29’) against it. We demonstrated the possibility to detect DPP6-expressing cells by single-photon emission computed tomography (SPECT)/ computed tomography (CT), but the correlation between the number of cells grafted and the SPECT signal was not assessed. Here, we investigate whether the 4hD29 nanobody allows us to detect different amounts of human pancreatic islets implanted into immune-deficient mice. In addition, we also describe the adaptation of the probe for use with positron emission tomography (PET). DPP6 expression was assessed in human samples using tissue arrays and immunohistochemistry. The effect of the 4hD29 nanobody on cell death and glucose-stimulated insulin secretion was measured in EndoC-βH1 cells and in human islets using Hoechst/propidium iodide staining and an anti-insulin ELISA, respectively. We performed in vivo SPECT imaging on severe combined immunodeficient (SCID) mice transplanted with different amounts of EndoC-βH1 cells (2 × 106, 5 × 106 and 10 × 106 cells), human islets (1000 and 3000) or pancreatic exocrine tissue using 99mTc-labelled 4hD29 nanobody. This DPP6 nanobody was also conjugated to N-chlorosuccinimide (NCS)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), radiolabelled with either 67Ga (SPECT) or 68Ga (PET) and used in a proof-of-principle experiment to detect DPP6-expressing cells (Kelly neuroblastoma) grafted in SCID mice. The DPP6 protein is mainly expressed in pancreatic islets. Importantly, the anti-DPP6 nanobody 4hD29 allows non-invasive detection of high amounts of EndoC-βH1 cells or human islets grafted in immunodeficient mice. This suggests that the probe must be further improved to detect lower numbers of islet cells. The 4hD29 nanobody neither affected beta cell viability nor altered insulin secretion in EndoC-βH1 cells and human islets. The conversion of 4hD29 nanobody into a PET probe was successful and did not alter its specificity. These findings suggest that the anti-DPP6 4hD29 nanobody may become a useful tool for the quantification of human islet grafts in mice and, pending future development, islet mass in individuals with diabetes.
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a nanobody based tracer targeting DPP6 for non invasive imaging of human pancreatic endocrine cells
Scientific Reports, 2017Co-Authors: Alexander Balhuizen, Sam Massa, Iris Mathijs, Jean Valery Turatsinze, Stephane Demine, Catarina Xavier, Olatz Villate, Isabelle Millard, Dominique Egrise, Carmen CapitoAbstract:There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-βH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells.
Erich Wettwer - One of the best experts on this subject based on the ideXlab platform.
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Interaction of DPP10a with Kv4.3 channel complex results in a sustained current component of human transient outward current Ito
Basic Research in Cardiology, 2015Co-Authors: K. Turnow, Diego Cotella, Ursula Ravens, Erich Wettwer, K. Metzner, M. J. Morales, M. Schaefer, T. Christ, S. KämmererAbstract:The sustained component of the K+ outward current in human atrial myocytes is believed to be due to the slowly inactivating ultra-rapid potassium current I Kur and not to the fast inactivating transient outward current I to. Here we provide evidence for contribution of I to to this late current due to the effects of dipeptidyl peptidase-like protein (DPP) 10 (DPP10a) interacting with Kv4.3 channels. We studied the late current component of I to in human atrial myocytes and CHO cells co-expressing Kv4.3 or Kv4.3/KChIP2 (control) and DPP proteins using voltage-clamp technique and a pharmacological approach. A voltage dependent and slowly inactivating late current (43 % of peak amplitude) could be observed in atrial myocytes. We found a similar current in CHO cells expressing Kv4.3/KChIP2 + DPP10a, but not in cells co-expressing Kv4.3 + DPP or Kv4.3/KChIP2 + DPP6-S. Assuming that DPP10a influences atrial I to, we detected DPP10 expression of three alternatively spliced mRNAs, DPP10 protein and colocalization of Kv4.3 and DPP10 proteins in human atrial myocytes. DPP10a did not affect properties of expressed Kv1.5 excluding a contribution to the sustained I Kur in atrial cells. To test for the contribution of Kv4-based I to on sustained K+ outward currents in human atrial myocytes, we used 4-AP to block I Kur, in combination with Heteropoda toxin 2 to block Kv4 channels. We could clearly separate an I to fraction of about 19 % contributing to the late current in atrial myocytes. Thus, the interaction of DPP10a, expressed in human atrium, with Kv4.3 channels generates a sustained current component of I to, which may affect late repolarization phase of atrial action potentials.
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effects of mirp1 and DPP6 β subunits on the blockade induced by flecainide of kv4 3 kchip2 channels
British Journal of Pharmacology, 2009Co-Authors: Ursula Ravens, Erich Wettwer, S Radicke, Miguel Vaquero, Ricardo Caballero, Ricardo Gomez, Lucia Nunez, Juan Tamargo, Eva DelponAbstract:Background and purpose: The human cardiac transient outward potassium current (Ito) is believed to be composed of the pore-forming KV4.3 α-subunit, coassembled with modulatory β-subunits as KChIP2, MiRP1 and DPP6 proteins. β-Subunits can alter the pharmacological response of Ito; therefore, we analysed the effects of flecainide on KV4.3/KChIP2 channels coassembled with MiRP1 and/or DPP6 β-subunits. Experimental approach: Currents were recorded in Chinese hamster ovary cells stably expressing KV4.3/KChIP2 channels, and transiently transfected with either MiRP1, DPP6 or both, using the whole-cell patch-clamp technique. Key results: In control conditions, KV4.3/KChIP2/MiRP1 channels exhibited the slowest activation and inactivation kinetics and showed an ‘overshoot’ in the time course of recovery from inactivation. The midpoint values (Vh) of the activation and inactivation curves for KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/MiRP1/DPP6 channels were ≈10 mV more negative than Vh values for KV4.3/KChIP2 and KV4.3/KChIP2/MiRP1 channels. Flecainide (0.1–100 μM) produced a similar concentration-dependent blockade of total integrated current flow (IC50 ≈10 μM) in all the channel complexes. However, the IC50 values for peak current amplitude and inactivated channel block were significantly different. Flecainide shifted the Vh values of both the activation and inactivation curves to more negative potentials and apparently accelerated inactivation kinetics in all channels. Moreover, flecainide slowed recovery from inactivation in all the channel complexes and suppressed the ‘overshoot’ in KV4.3/KChIP2/MiRP1 channels. Conclusions and implications: Flecainide directly binds to the KV4.3 α-subunit when the channels are in the open and inactivated state and the presence of the β-subunits modulates the blockade by altering the gating function. British Journal of Pharmacology (2008) 154, 774–786; doi:10.1038/bjp.2008.134; published online 21 April 2008
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the transmembrane β subunits kcne1 kcne2 and DPP6 modify pharmacological effects of the antiarrhythmic agent tedisamil on the transient outward current ito
Naunyn-schmiedebergs Archives of Pharmacology, 2009Co-Authors: Susanne Radicke, Diego Cotella, Daniele Sblattero, Ursula Ravens, Claudio Santoro, Erich WettwerAbstract:Accessory β-subunits modulate the pharmacology of ion channel blockers. The aim was to investigate differences in effects of the antiarrhythmic agent and open-channel blocker tedisamil on transient outward current I to (Kv4.3) when coexpressed with β-subunits potassium voltage-gated channel, Isk-related family, member 1 (KCNE1), potassium voltage-gated channel, Isk-related family, member 2 (KCNE2), or dipeptidyl-aminopeptidase-like protein 6 (DPP6) which modulate I to kinetics. Tedisamil inhibited I to with IC50 values of 16 μM for Kv4.3+KChIP2, 11 μM in the presence of KCNE1, and 14 μM for KCNE2. Values were higher in the presence of DPP6 or DPP6+KCNE2 (35 and 26 μM). K d values of tedisamil binding and rate constants were not affected by KCNE or DPP6. I to kinetics were accelerated by KCNE and DPP6, inactivation to a larger extent with DPP6. Tedisamil did not affect activation time course but apparently accelerated inactivation in all channel subunit combinations tested. Deletion of the intracellular domain of KCNE2 or DPP6 resulted in slowing of kinetics and increased tedisamil sensitivity (IC50 4 and 7 μM). It is concluded that apparent effects of DPP6 and deletion mutants (KCNE2 and DPP6) are due to the acceleration or slowing effects of the β-subunits on I to kinetics.
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effects of mirp1 and DPP6 b subunits on the blockade induced by flecainide of kv4 3 kchip2 channels
2008Co-Authors: S Radicke, Ursula Ravens, Miguel Vaquero, Ricardo Caballero, Ricardo Gomez, Lucia Nunez, Juan Tamargo, Erich WettwerAbstract:Background and purpose: The human cardiac transient outward potassium current (Ito) is believed to be composed of the pore-forming KV4.3 a-subunit, coassembled with modulatory b-subunits as KChIP2, MiRP1 and DPP6 proteins. b-Subunits can alter the pharmacological response of Ito; therefore, we analysed the effects of flecainide on KV4.3/KChIP2 channels coassembled with MiRP1 and/or DPP6 b-subunits. Experimental approach: Currents were recorded in Chinese hamster ovary cells stably expressing KV4.3/KChIP2 channels, and transiently transfected with either MiRP1, DPP6 or both, using the whole-cell patch-clamp technique. Key results: In control conditions, KV4.3/KChIP2/MiRP1 channels exhibited the slowest activation and inactivation kinetics and showed an ‘overshoot’ in the time course of recovery from inactivation. The midpoint values (Vh) of the activation and inactivation curves for KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/MiRP1/DPP6 channels were E10 mV more negative than Vh values for KV4.3/KChIP2 and KV4.3/KChIP2/MiRP1 channels. Flecainide (0.1–100 mM) produced a similar concentrationdependent blockade of total integrated current flow (IC50 E10 mM) in all the channel complexes. However, the IC50 values for peak current amplitude and inactivated channel block were significantly different. Flecainide shifted the Vh values of both the activation and inactivation curves to more negative potentials and apparently accelerated inactivation kinetics in all channels. Moreover, flecainide slowed recovery from inactivation in all the channel complexes and suppressed the ‘overshoot’ in KV4.3/ KChIP2/MiRP1 channels. Conclusions and implications: Flecainide directly binds to the KV4.3 a-subunit when the channels are in the open and inactivated state and the presence of the b-subunits modulates the blockade by altering the gating function. British Journal of Pharmacology (2008) 154, 774–786; doi:10.1038/bjp.2008.134; published online 21 April 2008