This review explores the evolving landscape of G-protein-coupled receptor (GPCR)-based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand-binding properties, structural insights, and spectral diversity.
The diagnosis of Parkinson's disease (PD) still lacks objective diagnostic markers independent of clinical criteria. Cerebrospinal fluid (CSF) samples from 36 PD and 42 age‐matched control patients were subjected to inductively coupled plasma‐sector field mass spectrometry and a total of 28 different elements were quantified. Different machine learning algorithms were applied to the dataset to identify a discriminating set of elements yielding a novel biomarker signature. Using 19 stably detected elements, the extreme gradient tree boosting model showed the best performance in the discrimination of PD and control patients with high specificity and sensitivity (78.6% and 83.3%, respectively), re‐classifying the training data to 100%. The 10 times 10‐fold cross‐validation yielded a good area under the receiver operating characteristic curve of 0.83. Arsenic, magnesium, and selenium all showed significantly higher mean CSF levels in the PD group compared to the control group (p = 0.01, p = 0.04, and p = 0.03). Reducing the number of elements to a discriminating minimum, we identified an elemental cluster (Se, Fe, As, Ni, Mg, Sr), which most importantly contributed to the sample discrimination. Selenium was identified as the element with the highest impact within this cluster directly followed by iron. After prospective validation, this elemental fingerprint in the CSF could have the potential to be used as independent biomarker for the diagnosis of PD. Next to their value as a biomarker, these data also argue for a prominent role of these highly discriminating six elements in the pathogenesis of PD.
Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre‐synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre‐ and post‐synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre‐synaptic dopamine function remain unclear. Non‐invasive imaging techniques such as positron emission tomography have revealed impaired pre‐synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre‐synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15–20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre‐treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre‐synaptic dopaminergic neurons are not initiated following a single exposure to the drug.
Purines are metabolic building blocks essential for all living organisms on earth. De novo purine biosynthesis occurs in the brain and appears to play important roles in neural development. Phosphoribosyl formylglycinamidine synthase (FGAMS , also known as PFAS or FGARAT ), a core enzyme involved in the de novo synthesis of purines, may play alternative roles in viral pathogenesis. To date, no thorough investigation of the endogenous expression and localization of de novo purine biosynthetic enzymes has been conducted in human neurons or in virally infected cells. In this study, we characterized expression of FGAMS using multiple neuronal models. In differentiated human SH ‐SY 5Y neuroblastoma cells, primary rat hippocampal neurons, and in whole‐mouse brain sections, FGAMS immunoreactivity was distributed within the neuronal cytoplasm. FGAMS immunolabeling in vitro demonstrated extensive distribution throughout neuronal processes. To investigate potential changes in FGAMS expression and localization following viral infection, we infected cells with the human pathogen herpes simplex virus 1. In infected fibroblasts, FGAMS immunolabeling shifted from a diffuse cytoplasmic location to a mainly perinuclear localization by 12 h post‐infection. In contrast, in infected neurons, FGAMS localization showed no discernable changes in the localization of FGAMS immunoreactivity. There were no changes in total FGAMS protein levels in either cell type. Together, these data provide insight into potential purine biosynthetic mechanisms utilized within neurons during homeostasis as well as viral infection.
Cover Image for this Issue: doi: 10.1111/jnc.14169 . 相似文献
Cerebrospinal fluid (CSF) α‐synuclein (ASYN) levels are emerging as a possible biomarker in a number of neurodegenerative conditions; however, there has been little study of such levels in demyelinating conditions with neurodegeneration such as multiple sclerosis (MS). In this study, we aimed to assess CSF ASYN levels in MS spectrum [clinically isolated syndrome (CIS) and MS] patients and compare them to those obtained in control subjects with benign neurological conditions (BNC). We used a recently developed, ultra‐sensitive sandwich enzyme‐linked immunosorbent assay to measure and compare CSF ASYN levels in three categories of subjects: BNC (n = 38), CIS (n = 36) and MS [Relapsing Remitting (RRMS, n = 22) and Primary Progressive (PPMS, n = 15)]. We also performed secondary analyses, including relationship of CSF ASYN levels to aging, gender, presence of CSF oligoclonal bands (OB) and gadolinium (Gd)‐enhancing demyelinating lesions on T1‐weighted MRIs. CSF ASYN levels were found to be significantly lower in the CIS (78.2 ± 7.5 pg/mL), RRMS (76.8 ± 5.1 pg/mL), and PPMS (76.3 ± 6.7 pg/mL) groups compared to the BNC (125.7 ± 13.6 pg/mL) group. Secondary analyses did not reveal additional correlations. Our results suggest that in a cohort of CIS and MS patients, CSF ASYN levels are decreased, thus providing another possible link between MS and neurodegeneration. Future studies will need to be performed to confirm and extend these findings, to lead to a fuller understanding of the possible biological link between ASYN and MS.
Taste information from type III taste cells to gustatory neurons is thought to be transmitted via synapses. However, the molecular mechanisms underlying taste transduction through this pathway have not been fully elucidated. In this study, to identify molecules that participate in synaptic taste transduction, we investigated whether complexins (Cplxs), which play roles in regulating membrane fusion in synaptic vesicle exocytosis, were expressed in taste bud cells. Among four Cplx isoforms, strong expression of Cplx2 mRNA was detected in type III taste cells. To investigate the function of CPLX2 in taste transduction, we observed taste responses in CPLX2‐knockout mice. When assessed with electrophysiological and behavioral assays, taste responses to some sour stimuli in CPLX2‐knockout mice were significantly lower than those in wild‐type mice. These results suggested that CPLX2 participated in synaptic taste transduction from type III taste cells to gustatory neurons.
Multiple sclerosis (MS) is considered an autoimmune demyelinating disease of the CNS and myelin‐derived glycolipids are one of the targets of this autoimmune attack. In this study, we examined for the first time the plasma distribution of sulfatide isoforms. Sulfatides with long‐chain (C24 : 0 or C24 : 1) and short‐chain (C16 : 0 or C18 : 0) fatty acids were quantified in plasma of relapsing–remitting MS patients by ultra‐high‐performance liquid chromatography tandem mass spectrometry. We found that C18 : 0 and C24 : 1 sulfatide plasma levels positively correlated with the Expanded Disability Status Scale. C16/C18 : 0 and C16/C24 : 0 ratios also correlated with the age and the time since last relapse. Healthy women showed higher levels of C16 : 0 sulfatide than healthy men; however, this gender difference disappeared in MS patients. Our data underline the potential use of sulfatides as biomarkers in relapsing–remitting MS and points to a possible association with the higher susceptibility of women to develop MS.
We are very sad that the ISN lost its President Kazuhiro Ikenaka, Professor and Chairman at National Institute for Physiological Sciences (NIPS), Director of Okazaki Institute of Integrative Biology. JNeurochem published an Obituary to value his outstanding achievements: Akio Wanaka et al. (2019) OBITUARY Kazuhiro Ikenaka (1952‐2018). https://doi.org/10.1111/jnc.14679
Autonomic control of heart rate is mediated by cardioinhibitory parasympathetic cholinergic neurons located in the brainstem and stimulatory sympathetic noradrenergic neurons. During embryonic development the survival and cholinergic phenotype of brainstem autonomic neurons is promoted by brain‐derived neurotrophic factor (BDNF). We now provide evidence that BDNF regulates heart rate by a mechanism involving increased brainstem cardioinhibitory parasympathetic activity. Mice with a BDNF haploinsufficiency exhibit elevated resting heart rate, and infusion of BDNF intracerebroventricularly reduces heart rate in both wild‐type and BDNF+/? mice. The atropine‐induced elevation of heart rate is diminished in BDNF+/? mice and is restored by BDNF infusion, whereas the atenolol‐induced decrease in heart rate is unaffected by BDNF levels, suggesting that BDNF signaling enhances parasympathetic tone which is diminished with BDNF haploinsufficiency. Whole‐cell recordings from pre‐motor cholinergic cardioinhibitory vagal neurons in the nucleus ambiguus indicate that BDNF haploinsufficiency reduces cardioinhibitory vagal neuron activity by increased inhibitory GABAergic and diminished excitatory glutamatergic neurotransmission to these neurons. Our findings reveal a previously unknown role for BDNF in the control of heart rate by a mechanism involving increased activation of brainstem cholinergic parasympathetic neurons
Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β‐alanine as a substrate. Proteoliposomes containing purified VGAT transport β‐alanine using Δψ but not ΔpH as a driving force. The Δψ‐driven β‐alanine uptake requires Cl?. VGAT also facilitates Cl? uptake in the presence of β‐alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β‐alanine uptake. These findings indicated that VGAT transports β‐alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β‐alanine transporter.
Cellular prion protein (PrPC ) is widely expressed and displays a variety of well‐described functions in the central nervous system (CNS ). Mutations of the PRNP gene are known to promote genetic human spongiform encephalopathies, but the components of gain‐ or loss‐of‐function mutations to PrPC remain a matter for debate. Among the proteins described to interact with PrPC is Stress‐inducible protein 1 (STI 1), a co‐chaperonin that is secreted from astrocytes and triggers neuroprotection and neuritogenesis through its interaction with PrPC . In this work, we evaluated the impact of different PrPC pathogenic point mutations on signaling pathways induced by the STI 1‐PrPC interaction. We found that some of the pathogenic mutations evaluated herein induce partial or total disruption of neuritogenesis and neuroprotection mediated by mitogen‐activated protein kinase (MAPK )/extracellular signal‐regulated kinases 1 and 2 (ERK 1/2) and protein kinase A (PKA ) signaling triggered by STI 1‐PrPC engagement. A pathogenic mutant PrPC that lacked both neuroprotection and neuritogenesis activities fail to promote negative dominance upon wild‐type PrPC . Also, a STI 1‐α7‐nicotinic acetylcholine receptor‐dependent cellular signaling was present in a PrPC mutant that maintained both neuroprotection and neuritogenesis activities similar to what has been previously observed by wild‐type PrPC . These results point to a loss‐of‐function mechanism underlying the pathogenicity of PrPC mutations.
The psychostimulant amphetamine (AMPH) is frequently used to increase catecholamine levels in attention disorders and positron emission tomography imaging studies. Despite the fact that most radiotracers for positron emission tomography studies are characterized in non‐human primates (NHPs), data on regional differences of the effect of AMPH in NHPs are very limited. This study examined the impact of AMPH on extracellular dopamine (DA) levels in the medial prefrontal cortex and the caudate of NHPs using microdialysis. In addition to differences in magnitude, we observed striking differences in the temporal profile of extracellular DA levels between these regions that can likely be attributed to differences in the regulation of dopamine uptake and biosynthesis. The present data suggest that cortical DA levels may remain elevated longer than in the caudate which may contribute to the clinical profile of the actions of AMPH.
Precise quantification of extracellular glutamate concentrations upon neuronal activation is crucial for the understanding of brain function and neurological disorders. While optogenetics is an outstanding method for the correlation between distinct neurons and their role in circuitry and behavior, the electrochemically inactive nature of glutamate has proven challenging for recording upon optogenetic stimulations. This difficulty is due to the necessity for using enzyme‐coated microelectrodes and the risk for light‐induced artifacts. In this study, we establish a method for the combination of in vivo optogenetic stimulation with selective measurement of glutamate concentrations using enzyme‐coated multielectrode arrays and amperometry. The glutamatergic subthalamic nucleus (STN ), which is the main electrode target site in deep brain stimulation treatment of advanced Parkinson′s disease, has recently proven opotogenetically targetable in Pitx2‐Cre‐transgenic mice and was here used as model system. Upon stereotactic injection of viral Channelrhodopsin2‐eYFP constructs into the STN , amperometric recordings were performed at a range of optogenetic stimulation frequencies in the globus pallidus, the main STN target area, in anesthetized mice. Accurate quantification was enabled through a multi‐step analysis approach based on self‐referencing microelectrodes and repetition of the experimental protocol at two holding potentials, which allowed for the identification, isolation and removal of photoelectric and photoelectrochemical artifacts. This study advances the field of in vivo glutamate detection with combined optogenetics and amperometric recordings by providing a validated analysis framework for application in a wide variety of glutamate‐based approaches in neuroscience.
Environmental stimuli that signal food availability hold powerful sway over motivated behavior and promote feeding, in part, by activating the mesolimbic system. These food‐predictive cues evoke brief (phasic) changes in nucleus accumbens (NAc) dopamine concentration and in the activity of individual NAc neurons. Phasic fluctuations in mesolimbic signaling have been directly linked to goal‐directed behaviors, including behaviors elicited by food‐predictive cues. Food‐seeking behavior is also strongly influenced by physiological state (i.e., hunger vs. satiety). Ghrelin, a stomach hormone that crosses the blood‐brain barrier, is linked to the perception of hunger and drives food intake, including intake potentiated by environmental cues. Notwithstanding, whether ghrelin regulates phasic mesolimbic signaling evoked by food‐predictive stimuli is unknown. Here, rats underwent Pavlovian conditioning in which one cue predicted the delivery of rewarding food (CS+) and a second cue predicted nothing (CS?). After training, we measured the effect of ghrelin infused into the lateral ventricle (LV) on sub‐second fluctuations in NAc dopamine using fast‐scan cyclic voltammetry and individual NAc neuron activity using in vivo electrophysiology in separate groups of rats. LV ghrelin augmented both phasic dopamine and phasic increases in the activity of NAc neurons evoked by the CS+. Importantly, ghrelin did not affect the dopamine nor NAc neuron response to the CS?, suggesting that ghrelin selectively modulated mesolimbic signaling evoked by motivationally significant stimuli. These data demonstrate that ghrelin, a hunger signal linked to physiological state, can regulate cue‐evoked mesolimbic signals that underlie food‐directed behaviors.
Huntington's disease (HD) is one of many neurodegenerative diseases with reported alterations in brain iron homeostasis that may contribute to neuropathogenesis. Iron accumulation in the specific brain areas of neurodegeneration in HD has been proposed based on observations in post‐mortem tissue and magnetic resonance imaging studies. Altered magnetic resonance imaging signal within specific brain regions undergoing neurodegeneration has been consistently reported and interpreted as altered levels of brain iron. Biochemical studies using various techniques to measure iron species in human samples, mouse tissue, or in vitro has generated equivocal data to support such an association. Whether elevated brain iron occurs in HD, plays a significant contributing role in HD pathogenesis, or is a secondary effect remains currently unclear.
The mammalian target of rapamycin (mTOR) signalling cascade is involved in the intracellular regulation of protein synthesis, specifically for proteins involved in controlling neuronal morphology and facilitating synaptic plasticity. Research has revealed that the activity of the mTOR cascade is influenced by several extracellular and environmental factors that have been implicated in schizophrenia. Therefore, there is reason to believe that one of the downstream consequences of dysfunction or hypofunction of these factors in schizophrenia is disrupted mTOR signalling and hence impaired protein synthesis. This results in abnormal neurodevelopment and deficient synaptic plasticity, outcomes which could underlie some of the positive, negative and cognitive symptoms of schizophrenia. This review will discuss the functional roles of the mTOR cascade and present evidence in support of a novel mTOR‐based hypothesis of the neuropathology of schizophrenia.
An increase in tau acetylation at K274 and K281 and abnormal mitochondrial dynamics have been observed in the brains of Alzheimer's disease (AD) patients. Here, we constructed three types of tau plasmids, TauKQ (acetylated tau mutant, by mutating its K274/K281 into glutamine to mimic disease-associated lysine acetylation), TauKR (non-acetylated tau mutant, by mutating its K274/K281 into arginine), and TauWT (wild-type human full-length tau). By transfecting these tau plasmids in HEK293 cells, we found that TauWT and TauKR induced mitochondrial fusion by increasing the level of mitochondrial fusion proteins. Conversely, TauKQ induced mitochondrial fission by reducing mitochondrial fusion proteins, exacerbating mitochondrial dysfunction and apoptosis. BGP-15 ameliorated TauKQ-induced mitochondrial dysfunction and apoptosis by improving mitochondrial dynamics. Our findings suggest that acetylation of K274/281 represents an important post-translational modification site regulating mitochondrial dynamics, and that BGP-15 holds potential as a therapeutic agent for mitochondria-associated diseases such as AD.
The retinoids are a family of compounds that in nature are derived from vitamin A or pro‐vitamin A carotenoids. An essential part of the diet for mammals, vitamin A has long been known to be essential for many organ systems in the adult. More recently, however, they have been shown to be necessary for function of the brain and new discoveries point to a central role in processes ranging from neuroplasticity to neurogenesis. Acting in several regions of the central nervous system including the eye, hippocampus and hypothalamus, one common factor in its action is control of biological rhythms. This review summarizes the role of vitamin A in the brain; its action through the metabolite retinoic acid via specific nuclear receptors, and the regulation of its concentration through controlled synthesis and catabolism. The action of retinoic acid to regulate several rhythms in the brain and body, from circadian to seasonal, is then discussed to finish with the importance of retinoic acid in the regular pattern of sleep.
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