Intravital Ca2+ imaging of pancreatic β cell function after bariatric surgery

Bariatric surgery has long become an appropriate and common treatment for patients with severe obesity and many related conditions such as type 2 diabetes. However, the exact mechanism leading to improved metabolism shortly after surgery, most notably independent of weight loss, has not yet been fully elucidated. Akalestou's recent publication, "Intravital imaging of islet Ca2+ dynamics reveals enhanced β cell connectivity after bariatric surgery in mice," provides the first detailed insight into the progression of pancreatic islet function after bariatric surgery. By transplanting pancreatic islets equipped with a genetically encoded calcium indicator into the anterior chamber of the eye, improvements in Ca2+ dynamics and a more potent β cell network were observed over an extended time course after the surgical procedure. In the following sections, we will take the opportunity to briefly outline the association between bariatric surgery and diabetes, highlight the issue of anesthesia during intravital Ca2+ imaging, and finally comment on some biological relationships related to β cell function.Published versionFamiljen Erling-Perssons Stiftelse, Stichting af Jochnick Foundation, Diabetesfonden, European Research Council (ERC) “Advanced Grant” (EYELETS), SSF Stiftelsen f ̈or Strategisk Forskning (Rambidrag MED-X 2018), KI Fonder, Vetenskapsrådet (The Swedisch Research Council), Berth von Kantzows Stiftelse, Novo Nordisk Foundation

Die physiologische Rolle der axonalen Bifurkation von sensorischen DRG- Neuronen vermittelt durch Npr2-abhängige cGMP-Signaltransduktion

Studies on afferent axon projections from sensory dorsal root ganglia (DRG) neurons into the spinal cord have demonstrated that the growth cone splitting (bifurcation) of these axons at the dorsal root entry zone into two daughter branches, which elongate either in caudal or rostral direction, is controlled by a Npr2-mediated cGMP signaling cascade. The aim of the thesis was twofold with respect to this bifurcation-inducing cGMP signaling cascade: (1) the generation of a floxed Npr2 mouse model for behavioral studies in order to gain first in vivo insights into the physiological function of axon bifurcation at the dorsal root entry zone, and (2) the investigation of mechanisms that regulate Npr2 activity as well as the search for potential protein interactions of components of the cGMP signaling cascade. (1) The generation of a conditional Npr2 knockout mouse was necessary due to the fact that Npr2-mediated cGMP signaling also contributes to the development and function of other tissues such as bone by endochondral ossification and the meiotic arrest of oocytes. Therefore, the loss of Npr2 function had to be confined to DRG neurons in order to allow interpretable behavioral studies and to clarify whether the transmission of sensory stimuli by afferent axons is hampered by impaired axonal branching in the spinal cord. The Cre-loxP recombination system was used as a genetic tool and a transgenic mouse model was generated containing two loxP target sites flanking exon 17 and exon 18 of the Npr2 gene. Whereas a first attempt to generate a floxed Npr2 mouse resulted in a hypomorphic phenotype due to a splice site mutation, the second attempt succeeded with the creation of the intended conditional Npr2 mouse mutant. Using the Wnt1-Cre mouse as a driver for Cre expression, experimental animals were generated which displayed undisturbed bone development in combination with impaired axon bifurcation. First behavioral studies (Hargreaves test) have revealed that the acute pain perception is reduced in these animals due to the lack of axon bifurcation. In general, the floxed Npr2 mouse provides a valuable tool for the investigation of Npr2-related phenotypes and therefore should be instrumental in studies of bone development, oogenesis or other Npr2-mediated physiological processes. (2) In order to analyze the regulation of Npr2 activity, site-directed mutagenesis of Npr2 followed by subsequent determination of the receptor activity revealed that cGMP production by the guanylyl cyclase domain of the receptor depends on the phosphorylation of five phosphorylation sites (Ser-513, Thr-516, Ser-518, Ser-523 and Ser-526) within the kinase homology domain of Npr2. Furthermore, to ascertain whether the cGMP signaling pathway is engaged in cross-talk with other proteins or signaling pathways, the proximity-dependent biotin identification (BioID) labeling assay based on the promiscuous biotin ligase BirA* was applied to screen for potential interaction partners of Npr2 and cGKIα in cell lines. Thus, a number of Npr2-specifically and cGKIα- specifically purified proteins were identified by mass spectrometry. The in vitro data of the biotin labeling assay provided first information about new potential interactors of the cGMP signaling cascade. However, it remains for future studies to experimentally confirm the actual link to cGMP signaling and the participation in axon bifurcation.Untersuchungen an Axonen von Spinalganglienneuronen im Rückenmark haben gezeigt, dass deren axonale Gabelung (Bifurkation), die beim Einwachsen in das Rückenmark entsteht, von einer Npr2-abhängigen cGMP-Signaltransduktion kontrolliert wird. Dabei entstehen zwei Tochteraxone, die konträr zueinander in rostraler und kaudaler Richtung entlang der Rückenmarksegmente weiterwachsen. Fehlt ein Mitglied der cGMP-Signalkaskade, wächst das Hauptaxon ohne zu bifukieren in nur einer Richtung im Rückenmark weiter. Die Zielsetzung der Doktorarbeit umfasste zwei Schwerpunkte hinsichtlich dieser axonalen Verzweigung im Rückenmark: (1) die Herstellung einer gefloxten Npr2 Mausmutante für verhaltensbiologische Untersuchungen, um erste In-vivo- Erkenntnisse über die physiologische Funktion der axonalen Verzweigung zu gewinnen und (2) die Untersuchung von Regulationsmechanismen, die die Aktivität von Npr2 kontrollieren sowie die Suche nach weiteren Proteinen, die mit der cGMP-Signalkaskade interagieren und eine Rolle für die axonale Verzweigung spielen. (1) Die Erzeugung einer konditionellen Npr2-Mausmutante, bei der das Npr2 Gen nur in den sensorischen Neuronen deaktiviert wird, war notwendig, da auch das Längenwachstum von Knochen (enchondrale Ossifikation) und der meiotische Arrest von Oozyten durch Npr2-vermittelte cGMP- Signaltransduktion reguliert wird. Eine gefloxte Npr2 Mausmutante wurde erzeugt, bei der zwei loxP-Sequenzen das Exon 17 und Exon 18 des Npr2 Gens flankieren. Die erste erzeugte gefloxte Npr2-Mausmutante wies allerdings auf Grund von Punktmutationen im Bereich der Exon-Intron-Grenzen bereits einen hypomorphen Phänotyp auf. Erst mit einer mutationsfreien, gefloxten Npr2-Maus und mittels der Wnt1-Cre-Maus konnte eine konditionelle Npr2-defiziente Mausmutante mit normaler Knochenentwicklung, aber ohne Bifurkation der sensorischen Axone im Rückenmark, erzeugt werden. Erste verhaltensbiologische Untersuchungen (Hargreaves Test) haben gezeigt, dass die akute Schmerzwahrnehmung von Tieren mit axonaler Bifurkationsstörung im Rückenmark reduziert ist. Die erzeugte gefloxte Npr2-Mausmutante ist darüber hinaus ein geeignetes Mausmodell, mit dessen Hilfe die Funktion der Npr2-vermittelten cGMP-Signaltransduktion auch in anderen Npr2-abhängigen physiologischen Prozessen untersucht werden kann. (2) Die In-vitro-Aktivität von Npr2-Mutanten, die durch ortsgerichtete Mutagenese verändert wurden, hat gezeigt, dass die Phosphorylierung des Rezeptormoleküls für die enzymatische Aktivität von großer Bedeutung ist. Nur wenn fünf Phosphorylierungsstellen (Ser-513, Thr-516, Ser-518, Ser-523 und Ser-526) innerhalb der Kinase- Homologie-Domäne von Npr2 phosphoryliert sind, kann der Ligand CNP die Guanylatzyklase-Aktivität des Rezeptors und somit die cGMP-Produktion vollständig induzieren. Um mögliche Interaktionspartner der cGMP-Signalkaskade nachzuweisen, die an der Rezeptorregulation oder der Strukturveränderung des Zytoskeletts beteiligt sind, wurde eine Biotin-Markierungs-Analyse (BioID) basierend auf der promisken Biotin-Protein-Ligase BirA* etabliert. Mit Hilfe von BirA*-Hybridproteinen von Npr2 und cGKIα, Streptavidin-Aufreinigung und massenspektrometrischer Analyse wurden Proteine bestimmt, die spezifisch für Npr2 oder cGKIα nachgewiesen werden konnten und somit potentielle Interaktionspartner der cGMP-Signalkaskade sind

The Absence of Sensory Axon Bifurcation Affects Nociception and Termination Fields of Afferents in the Spinal Cord

A cGMP signaling cascade composed of C-type natriuretic peptide, the guanylyl cyclase receptor Npr2 and cGMP-dependent protein kinase I (cGKI) controls the bifurcation of sensory axons upon entering the spinal cord during embryonic development. However, the impact of axon bifurcation on sensory processing in adulthood remains poorly understood. To investigate the functional consequences of impaired axon bifurcation during adult stages we generated conditional mouse mutants of Npr2 and cGKI (Npr2fl/fl;Wnt1Cre and cGKIKO/fl;Wnt1Cre) that lack sensory axon bifurcation in the absence of additional phenotypes observed in the global knockout mice. Cholera toxin labeling in digits of the hind paw demonstrated an altered shape of sensory neuron termination fields in the spinal cord of conditional Npr2 mouse mutants. Behavioral testing of both sexes indicated that noxious heat sensation and nociception induced by chemical irritants are impaired in the mutants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are not affected. Recordings from C-fiber nociceptors in the hind limb skin showed that Npr2 function was not required to maintain normal heat sensitivity of peripheral nociceptors. Thus, the altered behavioral responses to noxious heat found in Npr2fl/fl;Wnt1Cre mice is not due to an impaired C-fiber function. Overall, these data point to a critical role of axonal bifurcation for the processing of pain induced by heat or chemical stimuli

The absence of sensory axon bifurcation affects nociception and termination fields of afferents in the spinal cord

A cGMP signaling cascade composed of C-type natriuretic peptide, the guanylyl cyclase receptor Npr2 and cGMP-dependent protein kinase I (cGKI) controls the bifurcation of sensory axons upon entering the spinal cord during embryonic development. However, the impact of axon bifurcation on sensory processing in adulthood remains poorly understood. To investigate the functional consequences of impaired axon bifurcation during adult stages we generated conditional mouse mutants of Npr2 and cGKI (Npr2fl/fl;Wnt1Cre and cGKIKO/fl;Wnt1Cre) that lack sensory axon bifurcation in the absence of additional phenotypes observed in the global knockout mice. Cholera toxin labeling in digits of the hind paw demonstrated an altered shape of sensory neuron termination fields in the spinal cord of conditional Npr2 mouse mutants. Behavioral testing of both sexes indicated that noxious heat sensation and nociception induced by chemical irritants are impaired in the mutants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are not affected. Recordings from C-fiber nociceptors in the hind limb skin showed that Npr2 function was not required to maintain normal heat sensitivity of peripheral nociceptors. Thus, the altered behavioral responses to noxious heat found in Npr2fl/fl;Wnt1Cre mice is not due to an impaired C-fiber function. Overall, these data point to a critical role of axonal bifurcation for the processing of pain induced by heat or chemical stimuli

Subthalamic nucleus deep brain stimulation with a multiple independent constant current-controlled device in Parkinson\u27s disease (INTREPID): a multicentre, double-blind, randomised, sham-controlled study.

BACKGROUND: Deep brain stimulation (DBS) of the subthalamic nucleus is an established therapeutic option for managing motor symptoms of Parkinson\u27s disease. We conducted a double-blind, sham-controlled, randomised controlled trial to assess subthalamic nucleus DBS, with a novel multiple independent contact current-controlled (MICC) device, in patients with Parkinson\u27s disease. METHODS: This trial took place at 23 implanting centres in the USA. Key inclusion criteria were age between 22 and 75 years, a diagnosis of idiopathic Parkinson\u27s disease with over 5 years of motor symptoms, and stable use of anti-parkinsonian medications for 28 days before consent. Patients who passed screening criteria were implanted with the DBS device bilaterally in the subthalamic nucleus. Patients were randomly assigned in a 3:1 ratio to receive either active therapeutic stimulation settings (active group) or subtherapeutic stimulation settings (control group) for the 3-month blinded period. Randomisation took place with a computer-generated data capture system using a pre-generated randomisation table, stratified by site with random permuted blocks. During the 3-month blinded period, both patients and the assessors were masked to the treatment group while the unmasked programmer was responsible for programming and optimisation of device settings. The primary outcome was the difference in mean change from baseline visit to 3 months post-randomisation between the active and control groups in the mean number of waking hours per day with good symptom control and no troublesome dyskinesias, with no increase in anti-parkinsonian medications. Upon completion of the blinded phase, all patients received active treatment in the open-label period for up to 5 years. Primary and secondary outcomes were analysed by intention to treat. All patients who provided informed consent were included in the safety analysis. The open-label phase is ongoing with no new enrolment, and current findings are based on the prespecified interim analysis of the first 160 randomly assigned patients. The study is registered with ClinicalTrials.gov, NCT01839396. FINDINGS: Between May 17, 2013, and Nov 30, 2017, 313 patients were enrolled across 23 sites. Of these 313 patients, 196 (63%) received the DBS implant and 191 (61%) were randomly assigned. Of the 160 patients included in the interim analysis, 121 (76%) were randomly assigned to the active group and 39 (24%) to the control group. The difference in mean change from the baseline visit (post-implant) to 3 months post-randomisation in increased ON time without troublesome dyskinesias between the active and control groups was 3·03 h (SD 4·52, 95% CI 1·3-4·7; p INTERPRETATION: This double-blind, sham-controlled, randomised controlled trial provides class I evidence of the safety and clinical efficacy of subthalamic nucleus DBS with a novel MICC device for the treatment of motor symptoms of Parkinson\u27s disease. Future trials are needed to investigate potential benefits of producing a more defined current field using MICC technology, and its effect on clinical outcomes. FUNDING: Boston Scientific

Subthalamic nucleus deep brain stimulation with a multiple independent constant current-controlled device in Parkinson's disease (INTREPID): a multicentre, double-blind, randomised, sham-controlled study

Deep brain stimulation (DBS) of the subthalamic nucleus is an established therapeutic option for managing motor symptoms of Parkinson's disease. We conducted a double-blind, sham-controlled, randomised controlled trial to assess subthalamic nucleus DBS, with a novel multiple independent contact current-controlled (MICC) device, in patients with Parkinson's disease. This trial took place at 23 implanting centres in the USA. Key inclusion criteria were age between 22 and 75 years, a diagnosis of idiopathic Parkinson's disease with over 5 years of motor symptoms, and stable use of anti-parkinsonian medications for 28 days before consent. Patients who passed screening criteria were implanted with the DBS device bilaterally in the subthalamic nucleus. Patients were randomly assigned in a 3:1 ratio to receive either active therapeutic stimulation settings (active group) or subtherapeutic stimulation settings (control group) for the 3-month blinded period. Randomisation took place with a computer-generated data capture system using a pre-generated randomisation table, stratified by site with random permuted blocks. During the 3-month blinded period, both patients and the assessors were masked to the treatment group while the unmasked programmer was responsible for programming and optimisation of device settings. The primary outcome was the difference in mean change from baseline visit to 3 months post-randomisation between the active and control groups in the mean number of waking hours per day with good symptom control and no troublesome dyskinesias, with no increase in anti-parkinsonian medications. Upon completion of the blinded phase, all patients received active treatment in the open-label period for up to 5 years. Primary and secondary outcomes were analysed by intention to treat. All patients who provided informed consent were included in the safety analysis. The open-label phase is ongoing with no new enrolment, and current findings are based on the prespecified interim analysis of the first 160 randomly assigned patients. The study is registered with ClinicalTrials.gov, NCT01839396. Between May 17, 2013, and Nov 30, 2017, 313 patients were enrolled across 23 sites. Of these 313 patients, 196 (63%) received the DBS implant and 191 (61%) were randomly assigned. Of the 160 patients included in the interim analysis, 121 (76%) were randomly assigned to the active group and 39 (24%) to the control group. The difference in mean change from the baseline visit (post-implant) to 3 months post-randomisation in increased ON time without troublesome dyskinesias between the active and control groups was 3·03 h (SD 4·52, 95% CI 1·3–4·7; p<0·0001). 26 serious adverse events in 20 (13%) patients occurred during the 3-month blinded period. Of these, 18 events were reported in the active group and 8 in the control group. One death was reported among the 196 patients before randomisation, which was unrelated to the procedure, device, or stimulation. This double-blind, sham-controlled, randomised controlled trial provides class I evidence of the safety and clinical efficacy of subthalamic nucleus DBS with a novel MICC device for the treatment of motor symptoms of Parkinson's disease. Future trials are needed to investigate potential benefits of producing a more defined current field using MICC technology, and its effect on clinical outcomes. Boston Scientific