Membrane rafts and its GPI anchored complement inhibitors regulate insulin secretion

Type 2 diabetic patients usually exhibit an abnormal lipid profile. One such lipid that is constantly elevated in T2D is cholesterol. At the cellular level, cholesterol aids in tight packing of sphingolipids in certain regions of the plasma membrane and these specialized regions are termed ‘membrane rafts’, which act as signalling hubs. In pancreatic beta cells, these structures have been found to anchor important exocytotic proteins like VAMP2, syntaxin1A, SNAP25, Ca2+ channels and K+ channels and there are increasing number of evidences for its role in insulin secretory process. This thesis has aimed at unravelling the mechanisms by which membrane rafts regulate insulin exocytosis. Pancreatic islets from type 2 diabetic human donors show markedly decreased membrane raft staining intensity when compared to islets from healthy donors. Oxidation of membrane raft cholesterol using cholesterol oxidase (CO) significantly reduced membrane rafts, thereby disrupting its native structure and thus mimicking the diabetic condition. We have here shown that membrane raft disruption causes increased secretion of insulin under non-stimulatory conditions, depicting the loss of regulated insulin secretion. The increased basal insulin levels are accounted for by increased voltage-gated Ca2+ channel activity, which is further translated into increased influx of Ca2+ under basal conditions. Membrane rafts contain two important GPI anchored proteins, CD59 and CD55 which are crucial inhibitors of the complement system. Our results suggest that CD59 is the most highly expressed complement gene in human and rat pancreatic islets, the expression of which is downregulated in diabetic rodents. Intracellular CD59 is found colocalized with insulin, VAMP2 and syntaxin1A and this association is increased by high glucose incubations. Cells lacking CD59 exhibit decreased insulin exocytosis, which is caused by the lost CD59-mediated pairing of the SNARE proteins VAMP2 and syntaxin1A. CD59 thus plays an important role in insulin secretion. The extracellular CD59 is further observed to be important for maintaining the structural integrity of membrane rafts. CD55 is highly expressed in pancreatic islets and beta cells. Interestingly, in chronic hyperglycemia (diabetes), the expression of CD55 is further increased and is positively correlated with HbA1c. In fact, this is probably directly related to the hyperglycemic condition as glucose incubations regulate CD55 expression in human islets. In contrast to CD59, CD55 neither interacts with, nor regulates the insulin secretion machinery. However, CD55 shares the important capacity to regulate membrane rafts, which are disintegrated by silencing of CD55. Taken together, this thesis has shown that membrane rafts regulate insulin secretion by controlling the activity of voltage-gated Ca2+ channels. Furthermore, membrane raft marker protein and complement inhibitor CD59 is involved in the exocytosis process. This action is mediated by intracellular assistance in pairing of exocytosis-regulating SNARE proteins

Reactive Oxygen Species and Pressure Ulcer Formation after Traumatic Injury to Spinal Cord and Brain

Traumatic injuries to the nervous system, including the brain and spinal cord, lead to neurological dysfunction depending upon the severity of the injury. Due to the loss of motor (immobility) and sensory function (lack of sensation), spinal cord injury (SCI) and brain injury (TBI) patients may be bed-ridden and immobile for a very long-time. These conditions lead to secondary complications such as bladder/bowel dysfunction, the formation of pressure ulcers (PUs), bacterial infections, etc. PUs are chronic wounds that fail to heal or heal very slowly, may require multiple treatment modalities, and pose a risk to develop further complications, such as sepsis and amputation. This review discusses the role of oxidative stress and reactive oxygen species (ROS) in the formation of PUs in patients with TBI and SCI. Decades of research suggest that ROS may be key players in mediating the formation of PUs. ROS levels are increased due to the accumulation of activated macrophages and neutrophils. Excessive ROS production from these cells overwhelms intrinsic antioxidant mechanisms. While short-term and moderate increases in ROS regulate signal transduction of various bioactive molecules; long-term and excessively elevated ROS can cause secondary tissue damage and further debilitating complications. This review discusses the role of ROS in PU development after SCI and TBI. We also review the completed and ongoing clinical trials in the management of PUs after SCI and TBI using different technologies and treatments, including antioxidants

Autism Spectrum Disorders: A Recent Update on Targeting Inflammatory Pathways with Natural Anti-Inflammatory Agents

Autism spectrum disorder (ASD) is a heterogeneous category of developmental psychiatric disorders which is characterized by inadequate social interaction, less communication, and repetitive phenotype behavior. ASD is comorbid with various types of disorders. The reported prevalence is 1% in the United Kingdom, 1.5% in the United States, and ~0.2% in India at present. The natural anti-inflammatory agents on brain development are linked to interaction with many types of inflammatory pathways affected by genetic, epigenetic, and environmental variables. Inflammatory targeting pathways have already been linked to ASD. However, these routes are diluted, and new strategies are being developed in natural anti-inflammatory medicines to treat ASD. This review summarizes the numerous preclinical and clinical studies having potential protective effects and natural anti-inflammatory agents on the developing brain during pregnancy. Inflammation during pregnancy activates the maternal infection that likely leads to the development of neuropsychiatric disorders in the offspring. The inflammatory pathways have been an effective target for the subject of translational research studies on ASD

Complement inhibitor CD55 governs the integrity of membrane rafts in pancreatic beta cells, but plays no role in insulin secretion.

CD55 is a glycosylphosphatidylinositol-anchored protein, which inhibits complement activation by acting on the complement C3 convertases. CD55 is widely localized in the cholesterol rich regions of the cell plasma membrane termed membrane rafts. CD55 is attached to these specialized regions via a GPI link on the outer leaflet of the plasma membrane. Membrane rafts anchor many important signaling proteins, which control several cellular functions within the cell. For example, we recently demonstrated that the membrane raft protein and complement inhibitor CD59 also controls insulin secretion by an intracellular mechanism. Therefore, we have in this study aimed at addressing the expression and function of CD55 in pancreatic beta cells. To this end, we observe that CD55 is highly expressed in INS1 832/13 beta cells as well as human pancreatic islets. Diabetic human islets show a tendency for increased expression of CD55 when compared to the healthy controls. Importantly, silencing of CD55 in INS1 832/13 cells does not affect their insulin secretory capacity. On the other hand, silencing of CD55 diminished the intensity of membrane rafts as determined by Atto-SM staining. We hence conclude that CD55 expression is affected by glycemic status in human islets and plays a critical role in maintaining the conserved structure of rafts in pancreatic islets, which is similar to that of the related complement inhibitor CD59. However CD55 does not interfere with insulin secretion in beta cells, which is in sharp contrast to the action of the complement inhibitor CD59

Antagonistic L1 Adhesion Molecule Mimetic Compounds Inhibit Glioblastoma Cell Migration In Vitro

Cell adhesion molecule L1 is a cell surface glycoprotein that promotes neuronal cell migration, fosters regeneration after spinal cord injury and ameliorates the consequences of neuronal degeneration in mouse and zebrafish models. Counter-indicative features of L1 were found in tumor progression: the more L1 is expressed, the more tumor cells migrate and increase their metastatic potential. L1′s metastatic potential is further evidenced by its promotion of epithelial–mesenchymal transition, endothelial cell transcytosis and resistance to chemo- and radiotherapy. These unfortunate features are indicated by observations that cells that normally do not express L1 are induced to express it when becoming malignant. With the aim to ameliorate the devastating functions of L1 in tumors, we designed an alternative approach to counteract tumor cell migration. Libraries of small organic compounds were screened using the ELISA competition approach similar to the one that we used for identifying L1 agonistic mimetics. Whereas in the former approach, a function-triggering monoclonal antibody was used for screening libraries, we here used the function-inhibiting monoclonal antibody 324 that reduces the migration of neurons. We now show that the L1 antagonistic mimetics anagrelide, 2-hydroxy-5-fluoropyrimidine and mestranol inhibit the migration of cultured tumor cells in an L1-dependent manner, raising hopes for therapy

Single Nucleotide Polymorphism in Cell Adhesion Molecule L1 Affects Learning and Memory in a Mouse Model of Traumatic Brain Injury

The L1 cell adhesion molecule (L1) has demonstrated a range of beneficial effects in animal models of spinal cord injury, neurodegenerative disease, and ischemia; however, the role of L1 in TBI has not been fully examined. Mutations in the L1 gene affecting the extracellular domain of this type 1 transmembrane glycoprotein have been identified in patients with L1 syndrome. These patients suffer from hydrocephalus, MASA (mental retardation, adducted thumbs, shuffling gait, aphasia) symptoms, and corpus callosum agenesis. Clinicians have observed that recovery post-traumatic brain injury (TBI) varies among the population. This variability may be explained by the genetic differences present in the general population. In this study, we utilized a novel mouse model of L1 syndrome with a mutation at aspartic acid position 201 in the extracellular domain of L1 (L1-201). We assessed the impact of this specific single nucleotide polymorphism (SNP) localized to the X-chromosome L1 gene on recovery outcomes following TBI by comparing the L1-201 mouse mutants with their wild-type littermates. We demonstrate that male L1-201 mice exhibit significantly worse learning and memory outcomes in the Morris water maze after lateral fluid percussion (LFP) injury compared to male wild-type mice and a trend to worse motor function on the rotarod. However, no significant changes were observed in markers for inflammatory responses or apoptosis after TBI

Application of Antibodies to Neuronally Expressed Nogo-A Increases Neuronal Survival and Neurite Outgrowth

Nogo-A, a glycoprotein expressed in oligodendrocytes and central nervous system myelin, inhibits regeneration after injury. Antibodies against Nogo-A neutralize this inhibitory activity, improve locomotor recovery in spinal cord-injured adult mammals, and promote regrowth/sprouting/saving of damaged axons beyond the lesion site. Nogo-A is also expressed by neurons. Complete ablation of Nogo-A in all cell types expressing it has been found to lead to recovery in some studies but not in others. Neuronal ablation of Nogo-A reduces axonal regrowth after injury. In view of these findings, we hypothesized that, in addition to neutralizing Nogo-A in oligodendrocytes and myelin, Nogo-A antibodies may act directly on neuronal Nogo-A to trigger neurite outgrowth and neuronal survival. Here, we show that polyclonal and monoclonal antibodies against Nogo-A enhance neurite growth and survival of cultured cerebellar granule neurons and increase expression of the neurite outgrowth-promoting L1 cell adhesion molecule and polysialic acid. Application of inhibitors of signal transducing molecules, such as c-src, c-fyn, protein kinase A, and casein kinase II reduce antibody-triggered neurite outgrowth. These observations indicate that the recovery-promoting functions of antibodies against Nogo-A may not only be due to neutralizing Nogo-A in oligodendrocytes and myelin, but also to their interactions with Nogo-A on neurons

Eukaryotic translation initiation factor 3 subunit e controls intracellular calcium homeostasis by regulation of cav1.2 surface expression.

Inappropriate surface expression of voltage-gated Ca(2+)channels (CaV) in pancreatic ß-cells may contribute to the development of type 2 diabetes. First, failure to increase intracellular Ca(2+) concentrations at the sites of exocytosis impedes insulin release. Furthermore, excessive Ca(2+) influx may trigger cytotoxic effects. The regulation of surface expression of CaV channels in the pancreatic β-cells remains unknown. Here, we used real-time 3D confocal and TIRFM imaging, immunocytochemistry, cellular fractionation, immunoprecipitation and electrophysiology to study trafficking of L-type CaV1.2 channels upon β-cell stimulation. We found decreased surface expression of CaV1.2 and a corresponding reduction in L-type whole-cell Ca(2+) currents in insulin-secreting INS-1 832/13 cells upon protracted (15-30 min) stimulation. This internalization occurs by clathrin-dependent endocytosis and could be prevented by microtubule or dynamin inhibitors. eIF3e (Eukaryotic translation initiation factor 3 subunit E) is part of the protein translation initiation complex, but its effect on translation are modest and effects in ion channel trafficking have been suggested. The factor interacted with CaV1.2 and regulated CaV1.2 traffic bidirectionally. eIF3e silencing impaired CaV1.2 internalization, which resulted in an increased intracellular Ca(2+) load upon stimulation. These findings provide a mechanism for regulation of L-type CaV channel surface expression with consequences for β-cell calcium homeostasis, which will affect pancreatic β-cell function and insulin production

Elevated basal insulin secretion in type 2 diabetes caused by reduced plasma membrane cholesterol

Elevated basal insulin secretion under fasting conditions together with insufficient stimulated insulin release is an important hallmark of type 2 diabetes, but the mechanisms controlling basal insulin secretion remain unclear. Membrane rafts exist in pancreatic islet cells and spatially organize membrane ion channels and proteins controlling exocytosis, which may contribute to the regulation of insulin secretion. Membrane rafts (cholesterol and sphingolipid containing microdomains) were dramatically reduced in human type 2 diabetic and diabetic Goto-Kakizaki (GK) rat islets when compared with healthy islets. Oxidation of membrane cholesterol markedly reduced microdomain staining intensity in healthy human islets, but was without effect in type 2 diabetic islets. Intriguingly, oxidation of cholesterol affected glucose-stimulated insulin secretion only modestly, whereas basal insulin release was elevated. This was accompanied by increased intracellular Ca2+ spike frequency and Ca2+ influx and explained by enhanced single Ca2+ channel activity. These results suggest that the reduced presence of membrane rafts could contribute to the elevated basal insulin secretion seen in type 2 diabetes

The Complement Inhibitor CD59 Regulates Insulin Secretion by Modulating Exocytotic Events.

Type 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic β cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes