Recycling and EH domain proteins at the synapse

In neurons, a network of endocytic proteins accomplishes highly regulated processes such as synaptic vesicle cycling and the timely internalization of intracellular signaling molecules. In this review, we discuss recent advances on molecular networks created through interactions between proteins bearing the Eps15 homology (EH) domain and partner proteins containing the Asn–Pro–Phe (NPF) motif, which participate in important aspects of neuronal function as the synaptic vesicle cycle, the internalization of nerve growth factor (NGF), the determination of neuronal cell fate, the development of synapses and the trafficking of postsynaptic receptors. We discuss novel functional findings on the role of intersectin and synaptojanin and then we focus on the features of an emerging family of EH domain proteins termed EHDs (EH domain proteins), which are important for endocytic recycling of membrane proteins.     

SCAMP5 plays a critical role in axonal trafficking and synaptic localization of NHE6 to adjust quantal size at glutamatergic synapses

Glutamate uptake into synaptic vesicles (SVs) depends on cation/H⁺ exchange activity, which converts the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane at the presynaptic terminals. Thus, the proper recruitment of cation/H⁺ exchanger to SVs is important in determining glutamate quantal size, yet little is known about its localization mechanism. Here, we found that secretory carrier membrane protein 5 (SCAMP5) interacted with the cation/H⁺ exchanger NHE6, and this interaction regulated NHE6 recruitment to glutamatergic presynaptic terminals. Protein–protein interaction analysis with truncated constructs revealed that the 2/3 loop domain of SCAMP5 is directly associated with the C-terminal region of NHE6. The use of optical imaging and electrophysiological recording showed that small hairpin RNA–mediated knockdown (KD) of SCAMP5 or perturbation of SCAMP5/NHE6 interaction markedly inhibited axonal trafficking and the presynaptic localization of NHE6, leading to hyperacidification of SVs and a reduction in the quantal size of glutamate release. Knockout of NHE6 occluded the effect of SCAMP5 KD without causing additional defects. Together, our results reveal that as a key regulator of axonal trafficking and synaptic localization of NHE6, SCAMP5 could adjust presynaptic strength by regulating quantal size at glutamatergic synapses. Since both proteins are autism candidate genes, the reduced quantal size by interrupting their interaction may underscore synaptic dysfunction observed in autism.

The uptake of classical transmitters into synaptic vesicles (SVs) depends on a proton electrochemical gradient (ΔμH⁺), consisting of the chemical gradient (ΔpH) and membrane potential (Δψ) across the SV membrane (1). ΔμH⁺ is generated by vacuolar-type H⁺-ATPases and ion channels/transporters on SVs (2). There are five classes of vesicular neurotransmitter transporters on SVs, namely, vesicular glutamate transporters (VGLUT 1 to 3), vesicular GABA/glycine transporter (VGAT, VIAAT), vesicular monoamine transporters (VMAT 1, 2), vesicular acetylcholine transporter (VAChT), and vesicular nucleotide transporter (VNUT); these all use ΔpH and Δψ, but to different extents, to fill SVs with neurotransmitters (3). For example, transporters for the anionic neurotransmitter, glutamate, mainly utilize Δψ, whereas the transporters for the cationic neurotransmitter, acetylcholine, and zwitterionic neurotransmitters, GABA and glycine, depend on ΔpH and both ΔpH and Δψ, respectively (4). Therefore, homeostatic regulation of ΔμH⁺ is important for controlling the quantal size of neurotransmitter release.Cation/H⁺ exchange activity across the membrane is mostly attributed to monovalent Na⁺(K⁺)/H⁺ exchangers (NHEs) that present at the plasma membrane or intracellular organelles (5). In humans, the NHE superfamily comprises nine isoforms consisting of NHE1 to NHE5 on the plasma membrane, NHE6 and NHE9 on intracellular vesicles, and NHE7 and NHE8 on the Golgi apparatus (6). Plasma membrane isoforms recognize Na⁺ but not K⁺ and have important roles in the regulation of cytoplasmic pH, while the intracellular isoforms recognize K⁺ as well as Na⁺ (7), but their physiological roles remain poorly understood. Loss-of-function mutations of NHE6 and NHE9, the two endosomal subtypes (eNHEs), are implicated in multiple neurodevelopmental and neuropsychiatric disorders, including autism, Christianson syndrome, X-linked intellectual disability, and Angelman syndrome (8–13). NHE6 and NHE9 are highly expressed in the brain, including the hippocampus and cortex (14). Previous studies have found that SVs show an NHE activity that plays an important role in glutamate uptake into SVs by dissipating ΔpH and increasing Δψ (15, 16), and thus, eNHEs were considered to reside in SVs to perform this function (14). However, it was found that there was no defect in vesicular filling with glutamate or GABA in NHE9 knockout (KO) neurons, and NHE9 regulated the luminal pH of axonal endosomes rather than recycling SVs (17). In contrast, NHE6 was identified on SVs by using quantitative proteomics (18), and its presynaptic localization was shown by immunofluorescent analysis (19). In addition, more severe synaptic dysfunction was observed in NHE6 KO mice (19) than in NHE9 KO mice (17). These results suggest that NHE6 and NHE9 are not functionally redundant, and NHE6 is responsible for NHE activity in SVs that regulates glutamate uptake at presynaptic terminals. Evidently, the proper localization of NHE6 to SVs is of utmost importance in determining the glutamate quantal size, but the mechanism underlying NHE6 recruitment to SVs is mostly unknown.Secretory carrier membrane proteins (SCAMPs) are known to regulate vesicular trafficking and vesicle recycling processes. Of the five known SCAMPs, SCAMP1 and SCAMP5 are highly expressed in the brain and enriched in SVs (20). The study of SCAMP1 KO mice showed that it was not necessary for brain function and synaptic physiology (21), suggesting a critical role of SCAMP5 in synaptic function. Indeed, a recent genetic analysis showed that SCAMP5 was silenced on a derivative chromosome and was reduced in expression to ∼40% of normal in a patient with idiopathic, sporadic autism (22), and several SCAMP5 mutations reported in humans have been implicated in neurodevelopmental and neurodegenerative disorders such as intellectual disability, seizure, and Parkinson’s disease (23, 24). We also have provided evidence that SCAMP5 plays a critical role in SV endocytosis during strong neuronal activity (25) and the protein clearance at the presynaptic SV release site (26). These results suggest that SCAMP5 plays an important role in regulating the function and trafficking of synaptic proteins at presynaptic terminals.In this study, we showed that SCAMP5 directly interacted with NHE6. Optical imaging and electrophysiological recordings proved that when perturbing their interaction, axonal trafficking and synaptic localization of NHE6 were severely impaired, which subsequently lowered the resting luminal pH of SVs and reduced the amounts of glutamate release. Knockout of NHE6 occluded the effect of SCAMP5 knockdown (KD) without causing additional defects. Since NHE6 and SCAMP5 are candidate genes for autism (9, 22), the reduced quantal size following impairment of their interaction may relate to the synaptic dysfunction observed in autism.     

Neutrophils: Cinderella of innate immune system

Neutrophils are the first line of innate immune defense against infectious diseases. However, since their discovery by Elie Metchnikoff, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during acute infections. Now, extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Along with releasing various antimicrobial molecules, neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophils in the regulation of both innate and adaptive immunity during acute infectious or inflammatory conditions.     

Variations in Practice Patterns and Consistency With Published Guidelines for Balloon Aortic and Pulmonary Valvuloplasty: An Analysis of Data From the IMPACT Registry

Objectives

The authors sought to study variation in the practice of balloon aortic (BAV) and pulmonary valvuloplasty (BPV).

Resource reduction in pediatric chest pain: Standardized clinical assessment and management plan

Objective

Using a Standardized Clinical Assessment and Management Plan (SCAMP) for pediatric patients presenting to clinic with chest pain, we evaluated the cost impact associated with implementation of the care algorithm. Prior to introduction of the SCAMP, we analyzed charges for 406 patients with chest pain, seen in 2009, and predicted 21% reduction of overall charges had the SCAMP methodology been used. The SCAMP recommended an echocardiogram for history, examination, or ECG findings suggestive of a cardiac etiology for chest pain.

Design

Resource utilization was reviewed for 1517 patients (7‐21 years) enrolled in the SCAMP from July 2010 to April 2014.

Results

Compared to the 2009 historic cohort, patients evaluated by the SCAMP had higher rates of exertional chest pain (45% vs 37%) and positive family history (5% vs 1%). The SCAMP cohort had fewer abnormal physical examination findings (1% vs 6%) and abnormal electrocardiograms (3% vs 5%). Echocardiogram use increased in the SCAMP cohort compared to the 2009 historic cohort (45% vs 41%), whereas all other ancillary testing was reduced: exercise stress testing (4% SCAMP vs 28% historic), Holter (4% vs 7%), event monitors (3% vs 10%), and MRI (1% vs 2%). Total charges were reduced by 22% ($822 625) by use of the Chest Pain SCAMP, despite a higher percentage of patients for whom echocardiogram was recommended compared to the historic cohort.

Conclusions

The Chest Pain SCAMP effectively streamlines cardiac testing and reduces resource utilization. Further reductions can be made by algorithm refinement regarding echocardiograms for exertional symptoms.