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Lesson 4 Cell Signaling And Motility: G Proteins, Signal Transduction: Curations and Articles of reference as supplemental information: #TUBiol3373
Curator: Stephen J. Williams, Ph.D.
Updated 7/15/2019
Below please find the link to the Powerpoint presentation for lesson #4 for #TUBiol3373. The lesson first competes the discussion on G Protein Coupled Receptors, including how cells terminate cell signals. Included are mechanisms of receptor desensitization. Please NOTE that desensitization mechanisms like B arrestin decoupling of G proteins and receptor endocytosis occur after REPEATED and HIGH exposures to agonist. Hydrolysis of GTP of the alpha subunit of G proteins, removal of agonist, and the action of phosphodiesterase on the second messenger (cAMP or cGMP) is what results in the downslope of the effect curve, the termination of the signal after agonist-receptor interaction.
Stroke is a leading cause of death worldwide and the most common cause of long-term disability amongst adults, more particularly in patients with diabetes mellitus and arterial hypertension. Increasing evidence suggests that disordered physiological variables following acute ischaemic stroke, especially hyperglycaemia, adversely affect outcomes.
Post-stroke hyperglycaemia is common (up to 50% of patients) and may be rather prolonged, regardless of diabetes status. A substantial body of evidence has demonstrated that hyperglycaemia has a deleterious effect upon clinical and morphological stroke outcomes. Therefore, hyperglycaemia represents an attractive physiological target for acute stroke therapies.
However, whether intensive glycaemic manipulation positively influences the fate of ischaemic tissue remains unknown. One major adverse event of management of hyperglycaemia with insulin (either glucose-potassium-insulin infusions or intensive insulin therapy) is the occurrence of hypoglycaemia, which can also induce cerebral damage.
Doctors all over the world have debated whether intensive glucose management, which requires the use of IV insulin to bring blood sugar levels down to 80-130 mg/dL, or standard glucose control using insulin shots, which aims to get glucose below 180 mg/dL, lead to better outcomes after stroke.
A period of hyperglycemia is common, with elevated blood glucose in the periinfarct period consistently linked with poor outcome in patients with and without diabetes. The mechanisms that underlie this deleterious effect of dysglycemia on ischemic neuronal tissue remain to be established, although in vitro research, functional imaging, and animal work have provided clues.
While prompt correction of hyperglycemia can be achieved, trials of acute insulin administration in stroke and other critical care populations have been equivocal. Diabetes mellitus and hyperglycemia per se are associated with poor cerebrovascular health, both in terms of stroke risk and outcome thereafter.
Interventions to control blood sugar are available but evidence of cerebrovascular efficacy are lacking. In diabetes, glycemic control should be part of a global approach to vascular risk while in acute stroke, theoretical data suggest intervention to lower markedly elevated blood glucose may be of benefit, especially if thrombolysis is administered.
Both hypoglycaemia and hyperglycaemia may lead to further brain injury and clinical deterioration; that is the reason these conditions should be avoided after stroke. Yet, when correcting hyperglycaemia, great care should be taken not to switch the patient into hypoglycaemia, and subsequently aggressive insulin administration treatment should be avoided.
Early identification and prompt management of hyperglycaemia, especially in acute ischaemic stroke, is recommended. Although the appropriate level of blood glucose during acute stroke is still debated, a reasonable approach is to keep the patient in a mildly hyperglycaemic state, rather than risking hypoglycaemia, using continuous glucose monitoring.
The primary results from the Stroke Hyperglycemia Insulin Network Effort (SHINE) study, a large, multisite clinical study showed that intensive glucose management did not improve functional outcomes at 90 days after stroke compared to standard glucose therapy. In addition, intense glucose therapy increased the risk of very low blood glucose (hypoglycemia) and required a higher level of care such as increased supervision from nursing staff, compared to standard treatment.
Today’s lesson 3 explains how extracellular signals are transduced (transmitted) into the cell through receptors to produce an agonist-driven event (effect). This lesson focused on signal transduction from agonist through G proteins (GTPases), and eventually to the effectors of the signal transduction process. Agonists such as small molecules like neurotransmitters, hormones, nitric oxide were discussed however later lectures will discuss more in detail the large growth factor signalings which occur through receptor tyrosine kinases and the Ras family of G proteins as well as mechanosignaling through Rho and Rac family of G proteins.
Transducers: The Heterotrimeric G Proteins (GTPases)
An excellent review of heterotrimeric G Proteins found in the brain is given by
Cyclic AMP is an important second messenger. It forms, as shown, when the membrane enzyme adenylyl cyclase is activated (as indicated, by the alpha subunit of a G protein).
The cyclic AMP then goes on the activate specific proteins. Some ion channels, for example, are gated by cyclic AMP. But an especially important protein activated by cyclic AMP is protein kinase A, which goes on the phosphorylate certain cellular proteins. The scheme below shows how cyclic AMP activates protein kinase A.
Updated 7/15/2019
Additional New Studies on Regulation of the Beta 2 Adrenergic Receptor
We had discussed regulation of the G protein coupled beta 2 adrenergic receptor by the B-AR receptor kinase (BARK)/B arrestin system which uncouples and desensitizes the receptor from its G protein system. In an article by Xiangyu Liu in Science in 2019, the authors describe another type of allosteric modulation (this time a POSITIVE allosteric modulation) in the intracellular loop 2. See below:
Mechanism of β2AR regulation by an intracellular positive allosteric modulator
Xiangyu Liu1,*, Ali Masoudi2,*, Alem W. Kahsai2,*, Li-Yin Huang2, Biswaranjan Pani2, Dean P. Staus2, Paul J. Shim2, Kunio Hirata3,4, Rishabh K. Simhal2, Allison M. Schwalb2, Paula K. Rambarat2, Seungkirl Ahn2, Robert J. Lefkowitz2,5,6,†, Brian Kobilka1
Positive reinforcement in a GPCR
Many drug discovery efforts focus on G protein–coupled receptors (GPCRs), a class of receptors that regulate many physiological processes. An exemplar is the β2-adrenergic receptor (β2AR), which is targeted by both blockers and agonists to treat cardiovascular and respiratory diseases. Most GPCR drugs target the primary (orthosteric) ligand binding site, but binding at allosteric sites can modulate activation. Because such allosteric sites are less conserved, they could possibly be targeted more specifically. Liu et al. report the crystal structure of β2AR bound to both an orthosteric agonist and a positive allosteric modulator that increases receptor activity. The structure suggests why the modulator compound is selective for β2AR over the closely related β1AR. Furthermore, the structure reveals that the modulator acts by enhancing orthosteric agonist binding and stabilizing the active conformation of the receptor.
Abstract
Drugs targeting the orthosteric, primary binding site of G protein–coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β2– over the β1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.
Recent structures of GPCRs bound to allosteric modulators have revealed that receptor surfaces are decorated with diverse cavities and crevices that may serve as allosteric modulatory sites (1). This substantiates the notion that GPCRs are structurally plastic and can be modulated by a variety of allosteric ligands through distinct mechanisms (2-7). Most of these structures have been solved with negative allosteric modulators (NAMs), which stabilize receptors in their inactive states (1). To date, only a single structure of an active GPCR bound to a small-molecule positive allosteric modulator (PAM) has been reported, namely, the M2 muscarinic acetylcholine receptor with LY2119620 (8). Thus, mechanisms of PAMs and their potential binding sites remain largely unexplored.
Fig 1. Structure of the active state T4L-B2AR in complex with the orthosteric agonist BI-167107, nanobody 689, and compound 6FA. (A) The chemical structure of compound-6FA (Cmpd-6FA). (B) Isoproterenol (ISO) competition binding with 125I-cyanopindolol (CYP) to the β2AR reconstituted in nanodisks in the presence of vehicle (0.32% dimethylsulfoxide; DMSO), Cmpd-6, or Cmpd-6FA at 32 μM. Values were normalized to percentages of the maximal 125I-CYP binding level obtained from a one-site competition binding–log IC50 (median inhibitory concentration) curve fit. Binding curves were generated by GraphPad Prism. Points on curves represent mean ± SEM obtained from five independent experiments performed in duplicate. (C) Analysis of Cmpd-6FA interaction with the BI-167107–bound β2AR by ITC. Representative thermogram (inset) and binding isotherm, of three independent experiments, with the best titration curve fit are shown. Summary of thermodynamic parameters obtained by ITC: binding affinity (KD = 1.2 ± 0.1 μM), stoichiometry (N = 0.9 ± 0.1 sites), enthalpy (ΔH = 5.0 ± 1.2 kcal mol−1), and entropy (ΔS =13 ± 2.0 cal mol−1 deg−1). (D) Side view of T4L-β2AR bound to the orthosteric agonist BI-167107, nanobody 6B9 (Nb6B9), and Cmpd-6FA. The gray box indicates the membrane layer as defined by the OPM database. (E) Close-up view of Cmpd-6FA binding site. Covering Cmpd-6FA is 2Fo– Fc electron density contoured at 1.0 σ (green mesh).From Science 28 Jun 2019:
Vol. 364, Issue 6447, pp. 1283-1287
Fig 3. Fig. 3Mechanism of allosteric activation of the β2AR by Cmpd-6FA.
(A) Superposition of the inactive β2AR bound to the antagonist carazolol (PDB code: 2RH1) and the active β2AR bound to the agonist BI-167107, Cmpd-6FA, and Nb6B9. Close-up view of the Cmpd-6FA binding site is shown. The residues of the inactive (yellow) and active (blue) β2AR are depicted, and the hydrogen bond formed between Asp1303.49and Tyr141ICL2 in the active state is indicated by a black dashed line. (B) Topography of Cmpd-6FA binding surface on the active β2AR (left, blue) and the corresponding surface of the inactive β2AR (right, yellow) with Cmpd-6FA (orange sticks) docked on top. Molecular surfaces are of only those residues involved in interaction with Cmpd-6FA. Steric clash between Cmpd-6FA and the surface of inactive β2AR is represented by a purple asterisk. (C) Overlay of the β2AR bound to BI-167107, Nb6B9, and Cmpd-6FA with the β2AR–Gscomplex (PDB code: 3SN6). The inset shows the position of Phe139ICL2 relative to the α subunit of Gs. (D) Superposition of the active β2AR bound to the agonist BI-167107, Nb6B9, and Cmpd-6FA (blue) with the inactive β2AR bound to carazolol (yellow) (PDB code: 2RH1) as viewed from the cytoplasm. For clarity, Nb6B9 and the orthosteric ligands are omitted. The arrows indicate shifts in the intracellular ends of the TM helices 3, 5, and 6 upon activation and their relative distances.
Allosteric sites may not face the same evolutionary pressure as do orthosteric sites, and thus are more divergent across subtypes within a receptor family (24–26). Therefore, allosteric sites may provide a greater source of specificity for targeting GPCRs.
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D. Wacker, R. C. Stevens, B. L. Roth, How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170, 414–427 (2017).
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Additional information on Nitric Oxide as a Cellular Signal
Nitric oxide is actually a free radical and can react with other free radicals, resulting in a very short half life (only a few seconds) and so in the body is produced locally to its site of action (i.e. in endothelial cells surrounding the vascular smooth muscle, in nerve cells). In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980s he and others identified this substance as being NO.
Nitric oxide is implicated in many pathologic processes as well. Nitric oxide post translational modifications have been attributed to nitric oxide’s role in pathology however, although the general mechanism by which nitric oxide exerts its physiological effects is by stimulation of soluble guanylate cyclase to produce cGMP, these post translational modifications can act as a cellular signal as well. For more information of NO pathologic effects and how NO induced post translational modifications can act as a cellular signal see the following:
CABG: a Superior Revascularization Modality to PCI in Patients with poor LVF, Multivessel disease and Diabetes, Similar Risk of Stroke between 31 days and 5 years, post intervention
Reporter: Aviva Lev-Ari, PhD, RN
UPDATED on 9/4/2019
SYNTAX at 10 Years: Bypass vs PCI Still a Toss-Up Overall
But CABG beats stenting for important subgroups
SYNTAX was funded by Boston Scientific.
Thuijs, Stone, and Taggart disclosed no conflicts.
Baumbach reported receiving institutional research support from Abbott Vascular; and consultation and speaker fees from AstraZeneca, Sinomed, Microport, Abbott Vascular, Cardinal Health, KSH, Medtronic.
Pagano is the Secretary General of the European Association for Cardiothoracic Surgery.
LAST UPDATED
Overall, mortality curves did not diverge over time for SYNTAX participants now included in the trial’s extension study SYNTAXES: by 1o years, all-cause death reached 27.0% among those randomized to percutaneous coronary intervention (PCI) and 23.5% of the CABG arm (HR 1.17, 95% CI 0.97-1.41), according to Daniel Thuijs, MD, of Erasmus University Medical Centre in Rotterdam, the Netherlands.
However, people with three-vessel disease had particularly high mortality when assigned to PCI in lieu of CABG (27.7% vs 20.6%, HR 1.41, 95% CI 1.10-1.80), whereas peers with left main disease were no worse off after either procedure (26.1% vs 26.7%, HR 0.90, 95% CI 0.68-1.52),Thuijs told the audience during a late-breaking trial session at the European Society of Cardiology’s annual congress. The study was also published online in The Lancet.
CABG had a mortality benefit over PCI in patients with multivessel disease, particularly those with diabetes and higher coronary complexity. No benefit for CABG over PCI was seen in patients with left main disease. Longer follow-up is needed to better define mortality differences between the revascularisation strategies.
JACC Study, 7/2018
CONCLUSIONS
This individual patient-data pooled analysis demonstrates that 5-year stroke rates are significantly lower after PCI compared with CABG, driven by a reduced risk of stroke in the 30-day post-procedural period but a similar risk of stroke between 31 days and 5 years. The greater risk of stroke after CABG compared with PCI was confined to patients with multivessel disease and diabetes. Five-year mortality was markedly higher for patients experiencing a stroke within 30 days after revascularization.
European Journal of Cardiothoracic Surgery Study, 6/2018
CONCLUSIONS
Despite a longer length of hospital stay, patients with impaired LVF requiring intervention for coronary artery disease experienced a greater post-procedural survival benefit if they received CABG compared to PCI. We have demonstrated this at 30 days, 90 days, 1 year, 3 years, 5 years and 8 years following revascularization. At present, CABG remains a superior revascularization modality to PCI in patients with poor LVF.
New Studies on Clinical Outcomes from two Revascularization Strategies: CABG and PCI
J Am Coll Cardiol. 2018 Jul 24;72(4):386-398. doi: 10.1016/j.jacc.2018.04.071.
Stroke Rates Following Surgical Versus Percutaneous Coronary Revascularization.
Coronary artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) are used for coronary revascularization in patients with multivessel and left main coronary artery disease. Stroke is among the most feared complications of revascularization. Due to its infrequency, studies with large numbers of patients are required to detect differences in stroke rates between CABG and PCI.
OBJECTIVES:
This study sought to compare rates of stroke after CABG and PCI and the impact of procedural stroke on long-term mortality.
METHODS:
We performed a collaborative individual patient-data pooled analysis of 11 randomized clinical trials comparing CABG with PCI using stents; ERACI II (Argentine Randomized Study: Coronary Angioplasty With Stenting Versus Coronary Bypass Surgery in Patients With Multiple Vessel Disease) (n = 450), ARTS (Arterial Revascularization Therapy Study) (n = 1,205), MASS II (Medicine, Angioplasty, or Surgery Study) (n = 408), SoS (Stent or Surgery) trial (n = 988), SYNTAX (Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery) trial (n = 1,800), PRECOMBAT (Bypass Surgery Versus Angioplasty Using Sirolimus-Eluting Stent in Patients With Left Main Coronary Artery Disease) trial (n = 600), FREEDOM (Comparison of Two Treatments for Multivessel Coronary Artery Disease in Individuals With Diabetes) trial (n = 1,900), VA CARDS (Coronary Artery Revascularization in Diabetes) (n = 198), BEST (Bypass Surgery Versus Everolimus-Eluting Stent Implantation for Multivessel Coronary Artery Disease) (n = 880), NOBLE (Percutaneous Coronary Angioplasty Versus Coronary Artery Bypass Grafting in Treatment of Unprotected Left Main Stenosis) trial (n = 1,184), and EXCEL (Evaluation of Xience Versus Coronary Artery Bypass Surgery for Effectiveness of Left Main Revascularization) trial (n = 1,905). The 30-day and 5-year stroke rates were compared between CABG and PCI using a random effects Cox proportional hazards model, stratified by trial. The impact of stroke on 5-year mortality was explored.
RESULTS:
The analysis included 11,518 patients randomly assigned to PCI (n = 5,753) or CABG (n = 5,765) with a mean follow-up of 3.8 ± 1.4 years during which a total of 293 strokes occurred. At 30 days, the rate of stroke was 0.4% after PCI and 1.1% after CABG (hazard ratio [HR]: 0.33; 95% confidence interval [CI]: 0.20 to 0.53; p < 0.001). At 5-year follow-up, stroke remained significantly lower after PCI than after CABG (2.6% vs. 3.2%; HR: 0.77; 95% CI: 0.61 to 0.97; p = 0.027). Rates of stroke between 31 days and 5 years were comparable: 2.2% after PCI versus 2.1% after CABG (HR: 1.05; 95% CI: 0.80 to 1.38; p = 0.72). No significant interactions between treatment and baseline clinical or angiographic variables for the 5-year rate of stroke were present, except for diabetic patients (PCI: 2.6% vs. CABG: 4.9%) and nondiabetic patients (PCI: 2.6% vs. CABG: 2.4%) (p for interaction = 0.004). Patients who experienced a stroke within 30 days of the procedure had significantly higher 5-year mortality versus those without a stroke, both after PCI (45.7% vs. 11.1%, p < 0.001) and CABG (41.5% vs. 8.9%, p < 0.001).
CONCLUSIONS:
This individual patient-data pooled analysis demonstrates that 5-year stroke rates are significantly lower after PCI compared with CABG, driven by a reduced risk of stroke in the 30-day post-procedural period but a similar risk of stroke between 31 days and 5 years. The greater risk of stroke after CABG compared with PCI was confined to patients with multivessel disease and diabetes. Five-year mortality was markedly higher for patients experiencing a stroke within 30 days after revascularization.
Head SJ, Milojevic M, Daemen J, Ahn JM, Boersma E, Christiansen EH, Domanski MJ, Farkouh ME, Flather M, Fuster V, Hlatky MA, Holm NR, Hueb WA, Kamalesh M, Kim YH, Mäkikallio T, Mohr FW, Papageorgiou G, Park SJ, Rodriguez AE, Sabik JF, Stables RH, Stone GW, Serruys PW, Kappetein AP. Mortality after coronary artery bypass grafting versus percutaneous coronary intervention with stenting for coronary artery disease: a pooled analysis of individual patient data. Lancet. 2018 Feb 22 [Epub ahead of print]. doi: 10.1016/S0140-6736(18)30423-9. PMID: 29478841
Numerous randomised trials have compared coronary artery bypass grafting (CABG) with percutaneous coronary intervention (PCI) for patients with coronary artery disease. However, no studies have been powered to detect a difference in mortality between the revascularisation strategies.
Methods
We did a systematic review up to July 19, 2017, to identify randomised clinical trials comparing CABG with PCI using stents. Eligible studies included patients with multivessel or left main coronary artery disease who did not present with acute myocardial infarction, did PCI with stents (bare-metal or drug-eluting), and had more than 1 year of follow-up for all-cause mortality. In a collaborative, pooled analysis of individual patient data from the identified trials, we estimated all-cause mortality up to 5 years using Kaplan-Meier analyses and compared PCI with CABG using a random-effects Cox proportional-hazards model stratified by trial. Consistency of treatment effect was explored in subgroup analyses, with subgroups defined according to baseline clinical and anatomical characteristics.
Findings
We included 11 randomised trials involving 11 518 patients selected by heart teams who were assigned to PCI (n=5753) or to CABG (n=5765). 976 patients died over a mean follow-up of 3·8 years (SD 1·4). Mean Synergy between PCI with Taxus and Cardiac Surgery (SYNTAX) score was 26·0 (SD 9·5), with 1798 (22·1%) of 8138 patients having a SYNTAX score of 33 or higher. 5 year all-cause mortality was 11·2% after PCI and 9·2% after CABG (hazard ratio [HR] 1·20, 95% CI 1·06–1·37; p=0·0038). 5 year all-cause mortality was significantly different between the interventions in patients with multivessel disease (11·5% after PCI vs 8·9% after CABG; HR 1·28, 95% CI 1·09–1·49; p=0·0019), including in those with diabetes (15·5% vs 10·0%; 1·48, 1·19–1·84; p=0·0004), but not in those without diabetes (8·7% vs 8·0%; 1·08, 0·86–1·36; p=0·49). SYNTAX score had a significant effect on the difference between the interventions in multivessel disease. 5 year all-cause mortality was similar between the interventions in patients with left main disease (10·7% after PCI vs 10·5% after CABG; 1·07, 0·87–1·33; p=0·52), regardless of diabetes status and SYNTAX score.
Interpretation
CABG had a mortality benefit over PCI in patients with multivessel disease, particularly those with diabetes and higher coronary complexity. No benefit for CABG over PCI was seen in patients with left main disease. Longer follow-up is needed to better define mortality differences between the revascularisation strategies.
Comparison of the survival between coronary artery bypass graft surgery versus percutaneous coronary intervention in patients with poor left ventricular function (ejection fraction <30%): a propensity-matched analysis.
Existing evidence comparing the outcomes of coronary artery bypass graft (CABG) surgery versus percutaneous coronary intervention (PCI) in patients with poor left ventricular function (LVF) is sparse and flawed. This is largely due to patients with poor LVF being underrepresented in major research trials and the outdated nature of some studies that do not consider drug-eluting stent PCI.
METHODS:
Following strict inclusion criteria, 717 patients who underwent revascularization by CABG or PCI between 2002 and 2015 were enrolled. All patients had poor LVF (defined by ejection fraction <30%). By employing a propensity score analysis, 134 suitable matches (67 CABG and 67 PCI) were identified. Several outcomes were evaluated, in the matched population, using data extracted from national registry databases.
RESULTS:
CABG patients required a longer length of hospital stay post-revascularization compared to PCI in the propensity-matched population, 7 days (lower-upper quartile; 6-12) and 2 days (lower-upper quartile; 1-6), respectively (Mood’s median test, P = 0.001). Stratified Cox-regression proportional-hazards analysis of the propensity-matched population found that PCI patients experienced a higher adjusted 8-year mortality rate (hazard ratio 3.291, 95% confidence interval 1.776-6.101; P < 0.001). This trend was consistent amongst urgent cases of revascularization: patients with 3 or more vessels with coronary artery disease and patients where complete revascularization was achieved. Although sub-analyses found no difference between survival distributions of on-pump versus off-pump CABG (log-rank P = 0.726), both modes of CABG were superior to PCI (stratified log-rank P = 0.002).
CONCLUSIONS:
Despite a longer length of hospital stay, patients with impaired LVF requiring intervention for coronary artery disease experienced a greater post-procedural survival benefit if they received CABG compared to PCI. We have demonstrated this at 30 days, 90 days, 1 year, 3 years, 5 years and 8 years following revascularization. At present, CABG remains a superior revascularization modality to PCI in patients with poor LVF.
Using “Cerebral Organoids” to Trace the Elemental Composition of a Developing Brain
Curator: Marzan Khan, B.Sc
A research focused on the detection of micronutrient accumulation in the developing brain has been conducted recently by a team of scientific researchers in Brazil(1). Their study was comprised of a cutting-edge technology – human cerebral organoids, which are a close equivalent of the embryonic brain, in in-vitro models to identify some of the minerals essential during brain development using synchroton radiation(1). Since the majority of studies done on this matter have relied on samples from animal models, the adult brain or post-mortem tissue, this technique has been dubbed the “closest and most complete study system to date for understanding human neural development and its pathological manifestations”(2).
Cerebral organoids are three-dimensional miniature structures derived from human pluripotent stem cells that further differentiate into structures closely resembling the developing brain(2). Concentrating on two different time points during the developmental progression, the researchers illustrated the micronutrient content during an interval of high cell division marked on day 30 as well as day 40 when the organoids were starting to become mature neurons that secrete neurotransmitters, arranging into layers and forming synapses(2).
Synchrotron radiation X-ray fluorescence (SR-XRF) spectroscopy was used to discern each type of element present(2). After an incident beam of X-ray was directed at the sample, each atom emitted a distinct photon signature(2). Phosphorus (P), Potassium (P), Sulphur (S), Calcium (Ca), Iron (Fe), and Zinc (Zn) were found to be present in the samples in significant concentrations(2). Manganese (Mn), Nickel (Ni) and Copper (Cu) were also detected, but in negligible amounts, and therefore tagged as “ultratrace” elements(2). The distribution of these minerals, their concentration as well as their occurrence in pairs were examined at each interval(2).
Phosphorus was discovered to be the most abundant element in the cerebral organoid samples(3). Between the two time points at 30 days (cell proliferation) and 45 days (neuronal maturation) there was a marked decrease in P content(2). Since phosphorus is a major component of nucleotides and phospholipids, this reduction was clarified as a shift from a stage of cell division that requires the production of DNA and phospholipids, to a migratory and differentiation phase(2). Potassium levels were maintained during both phases, substantiating its role in mitotic cell division as well as cell migration over long distances(2). Sulfur levels were reportedly high at 30 days and 45 days(2). It was hypothesized that this element was responsible for the patterning of the organoids(2). Calcium, known to control transcription factors involved in neuronal differentiation and survival were detected in the micromolar range, along with zinc and iron(2). Zinc commits the differentiation of pluripotent stem cells into neuronal cells and iron is necessary for neuronal tissue expansion(2).
The cells in an embryo start to differentiate very early on- the neural plate is formed on the 16th day of contraception, which further folds and bulges out to become the nervous system (containing the brain and spinal cord regions)(3). Nutrients obtained from the mother are the primary sources of diet and energy for a developing embryo to fully differentiate and specialize into different organs(2). Lack of proper nutrition in pregnant mothers has been linked to many neurodegenerative diseases occurring in their progeny(2). Spina bifida which is characterized by the incomplete development of the brain and spinal cord, is a classic example of maternal malnutrition(2,4). Paucity of minerals in the diet of pregnant women are known to hamper learning and memory in children(2). Even Schizophrenia, Parkinson’s and Huntington’s disease have been associated to malnourishment(2). By showing the different types of elements present in statistically significant concentrations in cerebral organoids, the results of this study underscore the necessity of a healthy nourishment available to mothers during pregnancy for optimal development of the fetal brain(2).
2.Sartore RC, Cardoso SC, Lages YVM, Paraguassu JM, Stelling MP, Madeiro da Costa RF, Guimaraes MZ, Pérez CA, Rehen SK.(2017)Trace elements during primordial plexiform network formation in human cerebral organoids. PeerJ 5:e2927https://doi.org/10.7717/peerj.292
Alpha-linolenic acid given as enteral or parenteral nutritional intervention against sensorimotor and cognitive deficits in a mouse model of ischemic stroke
• High level of disability remains a substantial problem for stroke.
• An emerging concept to support stroke recovery is nutritional support.
• We compared whether oral or i.v supplementation of the omega-3, alpha-linolenic acid (ALA) best support recovery from stroke.
• Both types of ALA supplementation improved spatial learning and memory after stroke.
• This supports therapeutic plans using nutritional support in ALA in recovery from stroke.
Stroke is a leading cause of disability and death worldwide. Numerous therapeutics applied acutely after stroke have failed to improve long-term clinical outcomes. An emerging direction is nutritional intervention with omega-3 polyunsaturated fatty acids acting as disease-modifying factors and targeting post-stroke disabilities. Our previous studies demonstrated that the omega-3 precursor, alpha-linolenic acid (ALA) administrated by injections or dietary supplementation reduces stroke damage by direct neuroprotection, and triggering brain artery vasodilatation and neuroplasticity. Successful translation of putative therapies will depend on demonstration of robust efficacy on common deficits resulting from stroke like loss of motor control and memory/learning. This study evaluated the value of ALA as adjunctive therapy for stroke recovery by comparing whether oral or intravenous supplementation of ALA best support recovery from ischemia. Motor and cognitive deficits were assessed using rotarod, pole and Morris water maze tests. ALA supplementation in diet was better than intravenous treatment in improving motor coordination, but this improvement was not due to a neuroprotective effect since infarct size was not reduced. Both types of ALA supplementation improved spatial learning and memory after stroke. This cognitive improvement correlated with higher survival of hippocampal neurons. These results support clinical investigation establishing therapeutic plans using ALA supplementation
A research team led by scientists at SUNY Downstate Medical Center has identified a brain receptor that appears to initiate adolescent synaptic pruning, a process believed necessary for learning, but one that appears to go awry in both autism and schizophrenia.
Sheryl Smith, Ph.D., professor of physiology and pharmacology at SUNY Downstate, explained that “Memories are formed at structures in the brain known as dendritic spines that communicate with other brain cells through synapses. The number of brain connections decreases by half after puberty, a finding shown in many brain areas and for many species, including humans and rodents.”
This process is referred to as adolescent “synaptic pruning” and is thought to be important for normal learning in adulthood. Synaptic pruning is believed to remove unnecessary synaptic connections to make room for relevant new memories, but because it is disrupted in diseases such as autism and schizophrenia, there has recently been widespread interest in the subject.
“Our report is the first to identify the process which initiates synaptic pruning at puberty. Previous studies have shown that scavenging by the immune system cleans up the debris from these pruned connections, likely the final step in the pruning process,” added Dr. Smith. “Working with a mouse model we have shown that, at puberty, there is an increase in inhibitory GABA [gamma-aminobutyric acid] receptors, which are targets for brain chemicals that quiet down nerve cells. We now report that these GABA receptors trigger synaptic pruning at puberty in the mouse hippocampus, a brain area involved in learning and memory.”
The study (“Synaptic Pruning in the Female Hippocampus Is Triggered at Puberty by Extrasynaptic GABAA Receptors on Dendritic Spines”) is published online in eLife.
Dr. Smith noted that by reducing brain activity, these GABA receptors also reduce levels of a protein in the dendritic spine, kalirin-7, which stabilizes the scaffolding in the spine to maintain its structure. Mice that do not have these receptors maintain the same high level of brain connections throughout adolescence.
Dr. Smith pointed out that the mice with too many brain connections, which do not undergo synaptic pruning, are able to learn spatial locations, but are unable to relearn new locations after the initial learning, suggesting that too many brain connections may limit learning potential.
These findings may suggest new treatments targeting GABA receptors for “normalizing” synaptic pruning in diseases such as autism and schizophrenia, where synaptic pruning is abnormal. Research has suggested that children with autism may have an over-abundance of synapses in some parts of the brain. Other research suggests that prefrontal brain areas in persons with schizophrenia have fewer neural connections than the brains of those who do not have the condition.
Synaptic pruning in the female hippocampus is triggered at puberty by extrasynaptic GABAAreceptors on dendritic spines
JulieParato
Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, United States
No competing interests declared
</div>”>Sonia Afroz, JulieParato,
HuiShen
School of Biomedical Engineering, Tianjin Medical University, Tianjin, China
No competing interests declared
</div>”>HuiShen,
Sheryl SueSmith
Department of Physiology and Pharmacology, SUNY Downstate Medical Center, Brooklyn, United Statessheryl.smith@downstate.edu
Adolescent synaptic pruning is thought to enable optimal cognition because it is disrupted in certain neuropathologies, yet the initiator of this process is unknown. One factor not yet considered is the α4βδ GABAA receptor (GABAR), an extrasynaptic inhibitory receptor which first emerges on dendritic spines at puberty in female mice. Here we show that α4βδ GABARs trigger adolescent pruning. Spine density of CA1 hippocampal pyramidal cells decreased by half post-pubertally in female wild-type but not α4 KO mice. This effect was associated with decreased expression of kalirin-7 (Kal7), a spine protein which controls actin cytoskeleton remodeling. Kal7 decreased at puberty as a result of reduced NMDAR activation due to α4βδ-mediated inhibition. In the absence of this inhibition, Kal7 expression was unchanged at puberty. In the unpruned condition, spatial re-learning was impaired. These data suggest that pubertal pruning requires α4βδ GABARs. In their absence, pruning is prevented and cognition is not optimal.
Searches Related to Synaptic Pruning in the Female Hippocampus Is Triggered at Puberty by Extrasynaptic GABAA Receptors on Dendritic Spines
Researchers at Ruhr University Bochum (RUB; Germany) coupled nerve cell receptors to light-sensitive retinal pigments to understand how the serotonin neurotransmitter works and, therefore, learn what causes anxiety anddepression.
Prof. Dr. Olivia Masseck, who led the work, researches the causes of anxiety and depression. For more than 60 years, researchers have been hypothesising that the diseases are caused by, among other factors, changes to the level of serotonin. But understanding how the serotonin system works is quite difficult, says Masseck, who became junior professor for Super-Resolution Fluorescence Microscopy at RUB in April 2016.
With a method called optogenetics, Olivia Masseck (right) creates nerve cell receptors that are controllable with light. (Copyright: RUB, Damian Gorczany)
The number of receptors for serotonin in the brain amounts to 14, occurring in different cell types. Consequently, determining the functions that different receptors fulfill in the individual cell types is a complicated task. If, however, the proteins are coupled to light-sensitive pigments, they can be switched on and off with light of a specific color at high spatial and temporal precision. Masseck used this method, known as optogenetics, to characterize, for example, the properties of different light-sensitive proteins and identified the ones that are best suited as optogenetic tools. She has analyzed several light-sensitive varieties of the serotonin receptors 5-HT1A and 5-HT2C in great detail. Together with her collaborators, she has demonstrated in several studies that both receptors can control the anxiety behavior of mice.
To investigate the serotonin system more closely, Masseck and her research team is currently developing a sensor that is going to indicate the neurotransmitter in real time. One potential approach involves the integration of a modified form of a green fluorescent protein into a serotonin receptor.
In a brain slice, Olivia Masseck measures the activity of nerve cells in which she switches on their receptors using light stimulation. Via the pipette a red dye diffuses into the cell, rendering them visible in the brain slice. (Copyright: RUB, Damian Gorczany)
This protein produces green light only if it is embedded in a specific spatial structure. If a serotonin molecule binds to a receptor, the receptor changes its three-dimensional conformation. The objective is to integrate the fluorescent protein in the receptor so that its spatial structure changes together with that of the receptor when it binds a serotonin molecule, in such a way that the protein begins to glow.
New optogenetic tools by Julia Weiler
Anxiety and depression are two of the most frequently occurring mental disorders worldwide. Light-activated nerve cells may indicate how they are formed.
Statistically, every fifth individual suffers from depression or anxiety in the course of his or her life. The mechanisms that trigger these disorders are not yet fully understood, despite the fact that researchers have been studying the hypothesis that one of the underlying cause are changes to the level of the neurotransmitter serotonin for 60 years.
“Unfortunately, it is very difficult to understand how the serotonin system works,” says Prof Dr Olivia Masseck, who is junior professor for Super-Resolution Fluorescence Microscopy since the end of April 2016. She intends to fathom the mysteries of the complex system. The number of receptors for the neurotransmitter in the brain amounts to 14 in total, and they occur in different cell types. Consequently, determining the functions that different receptors fulfil in the individual cell types is a complicated task.
In order to fathom the purpose of such receptors, researchers used to observe which functions were inhibited after they had been activated or blocked with the aid of pharmaceutical drugs. However, many substances affect not just one receptor, but several at the same time. Moreover, researchers cannot tell receptors in the individual cell types apart when pharmaceutical drugs have been applied. “It had been impossible to study serotonin signalling pathways at high spatial and temporal resolution,” adds Masseck. Until the development of optogenetics.
“This method has revolutionised neuroscience,” says Olivia Masseck, whose collaborator Prof Dr Stefan Herlitze was one of the pioneers in this field. Optogenetics allows precise control over the activity of specific nerve cells or receptors with light. What sounds like science fiction, is routine at RUB’s Neuroscience Research Department. Masseck: “Until now, we had been passive observers, and monitoring cell activity was all we could do; now, we are able to manipulate it precisely.”
The researcher from Bochum is mainly interested in the 5-HT1A and 5-HT1B receptors, the so-called autoreceptors of the serotonin system. They occur in serotonin-producing cells, where they regulate the amount of released neurotransmitters; that means they determine the serotonin level in the brain.
Normally, 5-HT1A and 5-HT1B are activated when a serotonin molecule bonds to the receptor. The docking triggers a chain reaction in the cell. The effects of this signalling cascade include a reduced activity of the neural cell, which releases less neurotransmitter.
By modifying certain brain cells in the brains of mice, Olivia Masseck successfully activated the 5-HT1Areceptor without the aid of serotonin. She combined it with a visual pigment – so-called opsin. More specifically, she utilised blue or red visual pigments from the cones responsible for colour vision. This is how she generated a serotonin receptor that she could switch on with red or blue light. This method enables the RUB researcher to identify the role the 5-HT1A receptor plays in anxiety and depression.
To this end, she delivered the combined protein made up of light-sensitive opsin and serotonin receptor into the brain of mice using a virus that had been rendered harmless. Like a shuttle, it transports genetic information which contains the blueprint for the combined protein. Once injected into brain tissue, the virus implants the gene for the light-activated receptor in specific nerve cells. There, it is read, and the light-activated receptor is incorporated into the cell membrane.
The researcher was now able to switch the receptors on and off in a living mouse using light. She analysed in what way this manipulation affected the animals’ behaviour in an anxiety test, i.e. Open Field Test. For the purpose of the experiment, she placed individual mice in a large, empty Plexiglas box.
Under normal circumstances, the animals avoid the centre of the brightly-lit box, because it doesn’t offer any cover. Most of the time, they stay close to the walls. When Olivia Masseck switched on the 5-HT1Areceptor using light, the behaviour of the mice changed. They were less anxious and spent more time in the middle of the Plexiglas box.
These results were confirmed in a further test. Olivia Masseck stopped the time it took the mice to eat a food pellet in the middle of a large Plexiglas box. Normal animals waited between six and seven minutes before they ventured into the centre to feed. However, mice whose serotonin receptor was switched on started to feed after one or two minutes. “This is important evidence indicating that the 5-HT1A receptor signalling pathway in the serotonin system is linked to anxiety,” concludes Masseck.
In the next step, the researcher intends to find out in what way depressive behaviour is affected by the activation of the 5-HT1A receptor. “If the animals are exposed to chronic stress, they develop symptoms similar to those in humans with depression,” describes Masseck. “They might, for example, withdraw from social interactions.”
However, just like in humans, this applies to only a certain percentage of the mice. “Not every individual who suffers from chronic stress or experiences negative situations develops depression,” points out Masseck. What happens in the serotonin system of animals that are susceptible to depression, as opposed to that of animals that do not present any depressive symptoms? This is what the researcher intends to find out by deploying the optogenetic methods described above; in addition, she is currently developing a custom-built serotonin sensor.
Olivia Masseck’s assumption is that her findings regarding the neuronal circuits and molecular mechanisms of anxiety and depression are applicable to humans. Mice have similar cell functions, and their nervous system has a similar structure. The neuroscientist expects that optogenetics will one day be deployed in human applications.
“Genetic manipulation of cells for the purpose of controlling them with light might sound like science fiction,” she says, “but I am convinced that optogenetics will be used in human applications in the next decades.” It could, for example, be utilised for deep brain stimulation in Parkinson’s patients, because it facilitates precise activation of the required signalling pathways, with fewer side effects, at that.
“In the first step, optogenetics will be used in therapy of retinal diseases,” believes Olivia Masseck. Researchers are currently conducting experiments aiming at restoring the visual function in blind mice.
Olivia Masseck is aware that her research raises ethical questions. “We have to discuss in which applications we want or don’t want to use these techniques,” she says. Her research demonstrates how easily the lines between science-fiction films and scientific research can blur.
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