Expanding the genotype and phenotype spectrum of SYT1-associated neurodevelopmental disorder

PurposeSynaptotagmin-1 (SYT1) is a critical mediator of neurotransmitter release in the central nervous system. Previously reported missense SYT1 variants in the C2B domain are associated with severe intellectual disability, movement disorders, behavioral disturbances, and electroencephalogram abnormalities. In this study, we expand the genotypes and phenotypes and identify discriminating features of this disorder.MethodsWe describe 22 individuals with 15 de novo missense SYT1 variants. The evidence for pathogenicity is discussed, including the American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria, known structure–function relationships, and molecular dynamics simulations. Quantitative behavioral data for 14 cases were compared with other monogenic neurodevelopmental disorders.ResultsFour variants were located in the C2A domain with the remainder in the C2B domain. We classified 6 variants as pathogenic, 4 as likely pathogenic, and 5 as variants of uncertain significance. Prevalent clinical phenotypes included delayed developmental milestones, abnormal eye physiology, movement disorders, and sleep disturbances. Discriminating behavioral characteristics were severity of motor and communication impairment, presence of motor stereotypies, and mood instability.ConclusionNeurodevelopmental disorder–associated SYT1 variants extend beyond previously reported regions, and the phenotypic spectrum encompasses a broader range of severities than initially reported. This study guides the diagnosis and molecular understanding of this rare neurodevelopmental disorder and highlights a key role for SYT1 function in emotional regulation, motor control, and emergent cognitive function.     

Morphological changes in presynaptic terminals of the chick ciliary ganglion after stimulation in vivo

Summary The Edinger-Westphal nucleus of one day old chicks was stimulated in vivo. This nucleus projects via the oculomotor nerve to the ciliary ganglion. The stimulation produces morphological changes in the calyciform endings located in the ciliary ganglion. There is a significant reduction of the numerical density on area of the clear and the dense core vesicles. The numerical density of the coated vesicles is low compared to that of the clear vesicles. Their density is however almost doubled by the stimulation. The vesicles, the vacuoles and the plasma membrane were quantified using stereological procedures. A net loss of total membrane was found due to the loss of organelle membrane not compensated for by an equivalent increase of the plasma membrane. These observations are discussed in terms of the theory of vesicular membrane recycling as proposed by Heuser and Reese (1973).Supported by the Canadian Medical Research Council     

Purinergic transmission in the central nervous system

The adenosine 5′-triphosphate (ATP), discovered in 1929 by Karl Lohman, Cyrus Hartwell Fiske, and Yellagaprada SubbaRow, acts as an important extracellular signaling molecule. In the CNS, ATP can be released from synaptic terminals, either on its own or together with other neurotransmitters. After the release from the presynaptic terminals, ATP binds to a plethora of ionotropic and metabotropic receptors, which mediate its action as an excitatory neurotransmitter. Furthermore, ATP also acts as an important mediator in neuronal–glial communications because glial cells are endowed with numerous ATP receptors, which trigger Ca²⁺ signaling events and membrane currents in both macro and microglia. In addition, ATP can be released from astroglial cells, thereby acting as a mediator of glial–glial and glial–neuronal signaling.     

发动蛋白-1的脯氨酸和精氨酸富集域功能域与大鼠神经突触小体相互作用蛋白的筛选及鉴定

目的 筛选及鉴定发动蛋白-1(Dynamin-1)的脯氨酸和精氨酸富集域(PRD)功能域在大鼠神经突触小体中的相互作用蛋白.方法 构建Dynamin-1 PRD功能域的重组原核表达质粒pGEX-4T-2-PRD,通过大肠杆菌表达系统进行诱导表达;谷胱甘肽琼脂糖凝脂柱纯化得到融合蛋白GST-PRD,继而利用谷胱甘肽巯基转移酶沉淀技术,筛选出GST-PRD融合蛋白与分离提取的大鼠神经突触小体的相互作用蛋白,并通过液相质谱技术结合数据库,对上述获得的相互作用蛋白进行分析、鉴定.结果 通过构建pGEX-4T-2-PRD重组质粒及诱导表达,成功纯化出了融合蛋白GST-PRD;同时成功提取了大鼠神经突触小体,经谷胱甘肽巯基转移酶沉淀技术联合液相质谱分析,获得在大鼠神经突触小体中与Dynamin-1的PRD域有相互作用的蛋白共35个,分别属于突触囊泡相关蛋白、细胞骨架蛋白、代谢酶及其他类的蛋白.结论 本研究获得的与Dynamin-1的PRD域有相互作用的蛋白共35个.     

Agrin在神经系统损伤后修复中的作用

正神经系统主要由中枢神经系统(central nervous system,CNS)和周围神经系统(peripheral nervous systems,PNS)两大部分组成。CNS损伤包括脊髓损伤和脑神经损伤,主要以后者为主,具有发病率高、致残率高和死亡率高的特点。此外,CNS损伤后会引起包括DNA降解和细胞膜磷脂酰丝氨酸残基早期暴露在内的一系列程序性细胞死亡的病理过程,进     

Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system

Synaptic communication requires an efficient coupling of vesicle fusion to release neurotransmitter and vesicle retrieval to repopulate the synapse. In synapses of the CNS many proteins involved in exocytosis, endocytosis and refilling of vesicles have been identified. However, little is known about the organization and functioning of synaptic contacts in the enteric nervous system (ENS). We used fluorescent antibodies against presynaptic proteins (synaptobrevin, synaptophysin, synaptotagmin and bassoon) to identify synaptic contacts not only in guinea-pig enteric ganglia but also in the interconnecting fiber strands. Staining patterns were not altered by colchicine (100 μM), ruling out a contribution of protein transport at the time of fixation. Active release sites at fiber intersections and around neuronal cell bodies were labeled with FM1-43 (10 μM) by high K⁺ or electric field stimulation (EFS). During a second round of EFS, vesicles were reused, as reflected by dye loss. Destaining rates increased with stimulus frequency (2–30 Hz), reaching a maximum at about 15 Hz, likely caused by synaptic depression at higher frequencies. Tetrodotoxin (TTX, 1 μM) as well as nominally zero external Ca²⁺ (2 mM EGTA) prevented all destaining. The readily releasable pool (RRP, a subset of vesicles docked at the membrane and ready to fuse upon [Ca²⁺]_i increase) can be specifically released by a hypertonic challenge (500 mM sucrose). We measured this pool to be ∼27% of the total recycling pool, remarkably similar to synapses in the CNS. In whole-mount preparations, FM1-43 also reliably labeled active release sites in ganglia, fiber strands and in muscle bundles. The staining pattern indicated that the presynaptic antibodies mainly labeled active sites. The presence of numerous release sites suggests information processing capability within interconnecting fibers. With FM imaging, enteric synaptic function can be monitored independent of any postsynaptic modulation. Although electron microscopy data suggest that ENS synapses may not be as specialized as hippocampal synapses, remarkably similar release properties were measured.     

Exocytosis of secretory granules in the juxtaglomerular granular cells of kidneys

Background: There is little agreement as to the secretory process of renin granules in juxtaglomerular granular cells (JG cells) of kidneys, although a large number of studies of the regulation of renin secretion have been reported. Methods: The structural correlation between the stimuli and the secretory process was examined in mouse JG cells on renal cortical slice incubated with the beta-adrenergic agonist, isoproterenol; the loop diuretic, furocemide; the Ca²⁺ chelator, EGTA; and the actin filament-disrupting agent, cytochalasin B. Results and Conclusions: Treatment with isoproterenol (10⁻⁵−10⁻³ M) or furocemide (10⁻³M) in Ca²⁺-containing medium did not significantly affect the ultrastructure of JG cells. In slices incubated with isoproterenol or furocemide in the Ca²⁺-free medium, JG cells occasionally contained a few electron-lucent granules at the cell periphery in addition to the electrondense mature granules observed in the control slices. On rare occasions, the JG cells displayed omega-shaped cavities with electron-lucent matrices, a feature similar to the contents of electron-lucent granules. Cytochalasin B markedly promoted the effects of these stimulants in Ca²⁺-free medium. These findings suggest the participation of actin filament disassembly in the exocytotic process of the mature granules in JG cells. © 1995 Wiley-Liss, Inc.     

Neuromodulatory function of neuropeptides in the normal CNS

Neuropeptides are small protein molecules produced and released by discrete cell populations of the central and peripheral nervous systems through the regulated secretory pathway and acting on neural substrates. Inside the nerve cells, neuropeptides are selectively stored within large granular vesicles (LGVs), and commonly coexist in neurons with low-molecular-weight neurotransmitters (acetylcholine, amino acids, and catecholamines). Storage in LGVs is responsible for a relatively slow response to secretion that requires enhanced or repeated stimulation. Coexistence (i.e. the concurrent presence of a neuropeptide with other messenger molecules in individual neurons), and co-storage (i.e. the localization of two or more neuropeptides within individual LGVs in neurons) give rise to a complicated series of pre- and post-synaptic functional interactions with low-molecular-weight neurotransmitters.The typically slow response and action of neuropeptides as compared to fast-neurotransmitters such as excitatory/inhibitory amino acids and catecholamines is also due to the type of receptors that trigger neuropeptide actions onto target cells. Almost all neuropeptides act on G-protein coupled receptors that, upon ligand binding, activate an intracellular cascade of molecular enzymatic events, eventually leading to cellular responses. The latter occur in a time span (seconds or more) considerably longer (milliseconds) than that of low-molecular-weight fast-neurotransmitters, directly operating through ion channel receptors. As reviewed here, combined immunocytochemical visualization of neuropeptides and their receptors at the ultrastructural level and electrophysiological studies, have been fundamental to better unravel the role of neuropeptides in neuron-to-neuron communication.     

Mitochondrial uncoupling proteins in the central nervous system

Mitochondrial uncoupling proteins (UCPs), a subfamily of the mitochondrial transporter family, are related by sequence homology to UCP1. This protein, which is located in the inner mitochondrial membrane, dissipates the proton gradient between the intermembrane space and the mitochondrial matrix to uncouple electron transport from ATP synthesis. UCP1 (thermogenin) was first discovered in brown adipose tissue and is responsible for non-shivering thermogenesis. Expression of mRNA for three other UCP isoforms, UCP2, UCP4, and BMCP1/UCP5, has been found at high levels in brain. However, the physiological function(s) of UCPs in the brain have not been determined, although it has recently been postulated that UCPs regulate free radical flux from mitochondria by physiologically modulating mitochondrial membrane potential. In the CNS, this hypothesis has been studied primarily for UCP2. UCP2 message has been shown to be up-regulated in the CNS by stress signals such as kainate administration or ischemia, and overexpression of UCP2 has been reported to be neuroprotective against oxidative stress in vivo and in vitro, although the exact mechanism has not been fully established. In this review, studies on UCPs in the nervous system will be reviewed, and the potential roles of these intriguing proteins in acute and chronic diseases of the nervous system will be discussed.     

NSPc1相互作用蛋白的筛查及其与组蛋白乙酰化酶HBO1相互作用的验证

目的 寻找多梳家族(polycomb group)蛋白NSPc1的体内可能的相互作用蛋白.方法 通过酵母双杂交实验筛选与NSPc1相互作用的蛋白.使用Pull-down实验,Co-IP实验以及细胞内荧光共定位实验进一步证实双杂交中发现的相互作用.结果 分别以NSPc1蛋白的全长、N端和C端作为诱饵筛查人3月胎脑cDNA文库,发现N端诱饵可以与组蛋白乙酰化转移酶HBO1相互作用,该相互作用在体内外实验中都得到了验证.结论 组蛋白乙酰化酶HBO1是NSPc1的相互作用蛋白之一,该相互作用可能参与NSPc1对靶基因转录的表观抑制活性的发挥.     

极性蛋白与中枢神经系统发育

哺乳动物大脑神经元的形态多样性和突触连接的复杂性是极性细胞的典型例子,形成和维持神经元极性依赖多种极性蛋白的调节.从线虫受精卵发育到哺乳动物神经细胞的极性化通路中,许多极性蛋白存在进化保守机制.中枢神经系统发育的整个过程(包括神经元发生与移行、神经突生长以及突触联系的形成等)都有极性蛋白的直接或间接参与,是各种极性蛋白...     

Colony stimulating factors in the nervous system

Although traditionally seen as regulators of hematopoiesis, colony-stimulating factors (CSFs) have emerged as important players in the nervous system, both in health and disease. This review summarizes the cellular sources, patterns of expression and physiological roles of the macrophage (CSF-1, IL-34), granulocyte-macrophage (GM-CSF) and granulocyte (G-CSF) colony stimulating factors within the nervous system, with a particular focus on their actions on microglia. CSF-1 and IL-34, via the CSF-1R, are required for the development, proliferation and maintenance of essentially all CNS microglia in a temporal and regional specific manner. In contrast, in steady state, GM-CSF and G-CSF are mainly involved in regulation of microglial function. The alterations in expression of these growth factors and their receptors, that have been reported in several neurological diseases, are described and the outcomes of their therapeutic targeting in mouse models and humans are discussed.     

Heat shock proteins: Cellular and molecular mechanisms in the central nervous system

Emerging evidence indicates that heat shock proteins (HSPs) are critical regulators in normal neural physiological function as well as in cell stress responses. The functions of HSPs represent an enormous and diverse range of cellular activities, far beyond the originally identified roles in protein folding and chaperoning. HSPs are now understood to be involved in processes such as synaptic transmission, autophagy, ER stress response, protein kinase and cell death signaling. In addition, manipulation of HSPs has robust effects on the fate of cells in neurological injury and disease states. The ongoing exploration of multiple HSP superfamilies has underscored the pluripotent nature of HSPs in the cellular context, and has demanded the recent revamping of the nomenclature referring to these families to reflect a re-organization based on structure and function. In keeping with this re-organization, we first discuss the HSP superfamilies in terms of protein structure, regulation, expression and distribution in the brain. We then explore major cellular functions of HSPs that are relevant to neural physiological states, and from there we discuss known and proposed HSP impacts on major neurological disease states. This review article presents a three-part discussion on the array of HSP families relevant to neuronal tissue, their cellular functions, and the exploration of therapeutic targets of these proteins in the context of neurological diseases.     

Effects of restraint stress on the expression of proteins involved in synaptic vesicle exocytosis in the hippocampus

Chronic restraint stress has been associated with induction of morphological changes in the hippocampus. Postsynaptically, these changes include decreased length and branching of apical dendrites from CA3 pyramidal neurons, while presynaptically, depletion and clustering of synaptic vesicles have been observed. However, the molecular correlates of these changes remain poorly defined; while some studies have identified changes in the levels of some presynaptic proteins, none have assessed the coordinate expression of components of the membrane fusion complex, including synaptobrevin, syntaxin, and synaptosomal-associated protein 25 kDa, and their major regulatory molecules synaptotagmin, synaptophysin, and synapsin. Therefore, we undertook to assess the immunoreactivity of these proteins in hippocampal slices obtained from rats subjected to either acute (one 6 h session) or chronic (21 days at 6 h per day) of restraint stress. Specifically, we observed a significant increase in synaptobrevin immunoreactivity in the inner molecular layer of the dentate gyrus (54.2%; P=0.005), the stratum radiatum in the CA1 subfield (55.5%; P=0.007), and a region including the stratum lucidum and the proximal portion of the stratum radiatum in the CA3 subfield (52.7%; P=0.002); we also observed a trend toward increased synaptophysin levels in the stratum lucidum/radiatum of the CA3 subfield (8.0%; P=0.051) following chronic, but not acute, restraint stress. In that synaptobrevin has been associated with replenishment of the "readily-releasable" pool of synaptic vesicles and the efficiency of neurotransmitter release, the present results suggest that stress-induced changes in synaptobrevin may at least in part underlie the previously observed changes in synaptic and neuronal morphology.     

Nestin in central nervous system cells

This literature review reflects current knowledge on the intermediate filament protein nestin, which most authors regard as a marker of “neural stem/progenitor cells.” The structural-functional characteristics of nestin and its presence in various central nervous system cells at different stages of ontogenesis in normal and pathological conditions are discussed. __________ Translated from Morfologiya, Vol. 131, No. 1, pp. 85–90, January–February, 2007. Director: Corresponding Member of the Russian Academy of Medical Sciences Professor V. A. Otellin     

Synaptic and mitochondrial physiopathologic changes in the aging nervous system and the role of zinc ion homeostasis

Brain performances, e.g. learning and memory, decay during aging. Deterioration of synaptic junctions, as structural correlates of these key functions of the central nervous system, may play a central role in this impairment. Current research on the age-related changes of synapses is documenting that the numeric loss of contacts appears to trigger a compensatory reaction by the old CNS, i.e. the surviving junctional areas in old individuals are larger than in adult subjects. The final outcome of the balanced changes in synaptic number and size is that the overall synaptic junctional area per cubic micron of neuropil is also reduced in aging and this may account for the age-associated functional decay of CNS performances. Among the suggested determinants of synaptic deterioration in aging, a considerable number of recent studies support an early and pivotal role of the progressive decline of the mitochondrial metabolic competence, i.e. the capacity of select pools of organelles to provide adequate amounts of adenosine triphosphate. Quantitative ultrastructural studies together with cytochemistry of key enzymes of the respiratory chain (cytochrome oxidase and succinic dehydrogenase) have shown that mitochondrial dysfunctions play an early and central role in synaptic deterioration events associated with aging and neurodegenerative diseases. Among the various causes, the multiple mechanisms and molecules involved in zinc ion homeostasis have been supposed to be less efficient in the aging brain. Thus, a transient imbalance of free zinc ion concentration in the cytosol ([Zn2+]i) can be considered an unfavourable trigger of subtle mitochondrial damage and synaptic pathology.     

卵泡抑素样蛋白1在神经系统中的研究进展

卵泡抑素样蛋白1 (follistatin-like protein 1,FSTL1)是一种分泌型细胞外基质糖蛋白,在心血管疾病、自身免疫性疾病、肿瘤、炎症等疾病模型的发生、发展和转归过程中发挥重要作用,参与多种生物学过程,包括细胞增殖、凋亡、组织分化等.FSTL1在脊椎动物神经系统中调节神经胚形成和神经元分化诱导;参与大脑皮质进化机制和功能区域的划分;调节突触传递和维持躯体感觉正常阈值;减少脑卒中后神经元凋亡并改善神经缺陷功能.     

Accumulated glutamate levels in the synaptic vesicle are not maintained in the absence of active transport

We have investigated factors which may affect accumulated glutamate levels in synaptic vesicles and glutamate efflux. Agents which dissipate the electrochemical proton gradient resulted in a rapid reduction of steady-state vesicular glutamate levels, which was prevented by N-ethylmaleimide. Glutamate efflux was found to occur even in the presence of an electrochemical proton gradient, but was effectively inhibited by N-ethylmaleimide. These results suggest that accumulated glutamate levels in synaptic vesicles are not maintained unless glutamate is taken up continuously by an active transport mechanism, and they could provide an explanation for the lack of convincing evidence for the enrichment of endogenous glutamate in isolated synaptic vesicles.