Journal of Neurochemistry最新文献

Neurotransmitter:sodium symporters (NSS) reuptake neurotransmitter molecules from the synaptic space through Na⁺-coupled transport. They are thought to work via the alternating access mechanism, exploring multiple configurations dictated by the binding of substrates and ions. Much of the current knowledge about these transporters has been derived from examining the structure of the Leucine Transporter (LeuT), a bacterial counterpart to human NSSs. Multiple crystal structures of LeuT provided valuable information regarding the steps involved in this mechanism. Dynamical data connecting the crystal structure to the transport cycle are critical to understanding how ligands are translated through the membrane. In the present study, we applied ¹⁹F-based nuclear magnetic resonance (NMR) spectroscopy to ¹⁹F labelled LeuT to monitor how substrates and ions binding affect the conformations of the transporter. By selecting mutations and ligands known to affect the conformational equilibrium of LeuT, we identified and assigned four NMR resonances to specific conformational states of LeuT. We observe that Na⁺ ions produce closure of the extracellular vestibule to a state similarly induced by Na⁺ and substrates. Conversely, K⁺ ions seem to shift the conformational equilibrium toward inward-facing intermediates, arguably by competing with Na⁺. The present study assembles a framework for NMR-based dynamical studies of NSS transporters and demonstrates its feasibility for tackling large membrane LeuT-fold transporters.

Sleep is vital for maintaining physical and mental well-being, impacting cognitive functions like memory and learning through neuroplasticity. Sleep disturbances prevalent in neurological and psychiatric disorders exacerbate cognitive decline, imposing societal burdens. Exploring the relationship between sleep and neuroplasticity elucidates the mechanisms influencing cognition, particularly amidst the prevalent sleep disturbances in these clinical populations. While existing reviews provide valuable insights, gaps remain in understanding the neurophysiological mechanisms underlying sleep and cognitive function. This scoping review aims to investigate the characteristic patterns of sleep parameters and neurochemical biomarkers in reflecting neuroplasticity changes related to neurological and psychiatric disorders and to explore how these markers interact and influence cognition at the molecular level. Studies involving adults and older adults were included, excluding animal models and the paediatric population. Selected studies explored the relationship between sleep parameter or neurochemical biomarker changes and cognitive impairment, reflecting underlying neuroplasticity changes. Peer-reviewed articles, clinical trials, theses, and dissertations in English were included while excluding secondary research and non-peer-reviewed sources. A three-step search strategy was executed following the updated Joanna Briggs Institute methodology for scoping reviews. Published studies were retrieved from nine databases, grey literature, expert recommendations, and hand-searching of the included studies' bibliography. A basic qualitative content synthesis of 34 studies was conducted per JBI's scoping review guidance. Slow-wave and Rapid-Eye Movement sleep, sleep spindles, sleep cycle disruption, K-Complex(KC) density, Hippocampal sEEG, BDNF, IL-6, iNOS mRNA expression, plasma serotonin, CSF Aβ-42, t-tau and p-tau proteins, and serum cortisol revealed associations with cognitive dysfunction. Examining the relationship between sleep parameters, neurochemical biomarkers, and cognitive function reveals neuronal mechanisms that guide potential therapeutic interventions and enhance quality patient care.

Neuronal growth regulator 1 (NEGR1) is a synaptic plasma membrane localized cell adhesion molecule implicated in a wide spectrum of psychiatric disorders. By RNAseq analysis of the transcriptomic changes in the brain of NEGR1-deficient mice, we found that NEGR1 deficiency affects the expression of the Gad2 gene. We show that glutamic acid decarboxylase 65 (GAD65), the Gad2 - encoded enzyme synthesizing the inhibitory neurotransmitter GABA on synaptic vesicles, accumulates non-synaptically in brains of NEGR1-deficient mice. The density of non-synaptic GAD65 accumulations is also increased in NEGR1 deficient cultured hypothalamic neurons, and this effect is rescued by re-expression of NEGR1. By using a novel biosensor of the plasma membrane attachment of GAD65, we demonstrate that GAD65 attaches to the plasma membrane. NEGR1 promotes palmitoylation-dependent clearance of GAD65 from the plasma membrane and targeting of GAD65 to plasma membrane-derived endocytic vesicles. In NEGR1 deficient cultured hypothalamic neurons, the synaptic and extrasynaptic levels of the plasma membrane attached GAD65 are increased, and the synaptic levels of GABA are reduced. NEGR1-deficient mice are characterized by reduced body weight, lower GABAergic synapse densities in the arcuate nucleus, and blunted responsiveness to the reinforcing effects of food rewards. Our results indicate that abnormalities in synaptic GABA synthesis can contribute to brain disorders associated with abnormal expression of NEGR1 in humans.

The dopamine (DA) transporter (DAT) is a major determinant of DAergic neurotransmission, and is a primary target for addictive and therapeutic psychostimulants. Evidence accumulated over decades in cell lines and in vitro preparations revealed that DAT function is acutely regulated by membrane trafficking. Many of these findings have recently been validated in vivo and in situ, and several behavioral and physiological findings raise the possibility that regulated DAT trafficking may impact DA signaling and DA-dependent behaviors. Here we review key DAT trafficking findings across multiple systems, and discuss the cellular mechanisms that mediate DAT trafficking, as well as the endogenous receptors and signaling pathways that drive regulated DAT trafficking. We additionally discuss recent findings that DAT trafficking dysfunction correlates to perturbations in DA signaling and DA-dependent behaviors.

To date, several studies have integrated genome-wide association studies (GWAS) and expression quantitative trait loci (eQTL) data from bulk tissues to identify novel Alzheimer's disease (AD) genetic variants and susceptibility genes. However, there is highly cell-type-specific nature in different bulk eQTL data. Until now, eQTL data from different brain single cells have been reported. Therefore, integrating eQTL data from different brain single-cell types along with AD GWAS data makes biological sense for studying the potential biological explanations of AD. Here, we utilized the summary-data-based Mendelian randomization (SMR) method to integrate AD GWAS data with eQTL data from eight brain single-cell types. We identified a larger number of significant genes compared to previous SMR study based on bulk eQTL. Notably, microglia exhibited the highest number of significant genes. Moreover, we conducted validation-phase SMR analysis, single-cell analysis, protein–protein interaction (PPI), druggability evaluation, functional enrichment analyses, and colocalization analysis of the top 20 SMR significant genes in microglia. We found that most genes passed the validation and were significantly enriched in microglia. PPI analysis uncovered interactions among PICALM, BIN1, RIN3, CD2AP, CASS4, and MS4A6E. Five most significant SMR genes were further validated through colocalization analysis. RIN3 is the only significant gene across all mentioned analyses and is a novel AD susceptibility gene at the genome-wide significance level. Druggability evaluation identified KCNQ3, HLA-DQB1, and RABEP1 as known genes previously targeted for drug development in neurological disorders, suggesting their potential therapeutic relevance in AD.

Neurotransmitter transporters (NTTs) control synaptic responses by modulating the concentration of neurotransmitters at the synaptic cleft. Glutamate is the most abundant excitatory neurotransmitter in the brain and needs to be finely tuned in time and space to maintain a healthy brain and precise neurotransmission. The glutamate transporter EAAT2 (SLC1A2) is primarily responsible for glutamate clearance. EAAT2 impairment has been associated with Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both monogenic and sporadic forms of PD, of which the common substitution Gly2019Ser is associated with a significant deficit in EAAT2 expression. The role of pathological mutants of the LRRK2 is intensively studied and reviewed. Here we have focused the attention on the physiological role of LRRK2 on EAAT2, comparing the activity of NTTs with or without the LRRK2 kinase. By heterologous expression in Xenopus laevis oocytes and two-electrode voltage clamp, the current amplitudes of the selected NTTs and kinetic parameters have been collected in the presence and absence of LRRK2. The results show that EAAT2 expression and function are impaired in the absence of the kinase and also under its pharmacological inhibition via MLi-2 treatment. LRRK2 stabilizes EAAT2 expression increasing the amount of transporter at the plasma membrane. Interestingly, the LRRK2 action is EAAT2-specific, as we observed no significant changes in the transport current amplitude and kinetic parameters obtained for the other excitatory and inhibitory NTTs studied. This study, for the first time, demonstrates the physiological importance of LRRK2 in EAAT2 function, highlighting the specificity of LRRK2-mediated modulation of EAAT2 and suggesting a potential role for the kinase as a checkpoint for preserving neurons from excitotoxicity. In brain conditions associated with impaired glutamate clearance, targeting LRRK2 for EAAT2 regulation may offer novel therapeutic opportunities.

Peripheral myelin is synthesized by glial cells called Schwann cells (SCs). SC development and differentiation must be tightly regulated to avoid any pathological consequence affecting peripheral nerve function. Neuropathic symptoms can arise from developmental issues in SCs, as well as in adult life through processes affecting mature SCs. In this review we focus on SC differentiation from the immature towards the myelinating and non-myelinating SC stages, defining molecular mechanisms outlining radial sorting, a multi-stepped event essential for immature SC differentiation and myelination. We also describe mechanisms regulating myelin sheath maintenance and SC homeostasis during aging. Finally, we will conclude with some remaining questions in the field of SC biology.

2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, and its loss in mice and humans causes neurodegeneration. Additionally, CNPase is frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3′-hydrolysis of 2′,3′-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognising the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibres and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional studies on myelin formation, dynamics, and disease, including high-resolution imaging of nerve tissue.

Aging affects virtually all organs of the body, but perhaps it has the most profound effects on the brain and its neurotransmitter systems, which influence a wide range of crucial functions, such as attention, focus, mood, neuroendocrine and autonomic functions, and sleep cycles. All of these essential functions, as well as fundamental cognitive processes such as memory, recall, and processing speed, utilize neuronal circuits that depend on neurotransmitter signaling between neurons. Glutamate (Glu), the main excitatory neurotransmitter in the CNS, is involved in most neuronal excitatory functions, including release of the neurotransmitter norepinephrine (NE). Previous studies from our lab demonstrated that the age-associated decline in Glu-stimulated NE release in rat cerebral cortex and hippocampus mediated by NMDA glutamate receptors, as well as deficits in dendritic spines, and cognitive functions are fully rescued by the CNS stimulant amphetamine. Here we further investigated Glu-stimulated NE release in the cerebral cortex to identify additional novel target sites for restoration of Glu-stimulated NE release. We found that blockade of alpha-2 adrenergic receptors fully restores Glu-stimulated NE release to the levels of young controls. In addition, we investigated the density and responsiveness of NMDA receptors as a potential underlying neuronal mechanism that could account for the observed age-associated decline in Glu-stimulated NE release. In the basal state of the receptor (no added glutamate and glycine) the density of NMDA receptors in the cortex from young and aged rats was similar. However, in contrast, in the presence of 10 μM added glutamate, which opens the receptor channel and increases the number of available [³H]-MK-801 binding sites within the channel, the density of [³H]-MK-801 binding sites was significantly less in the cortex from aged rats.

Neuroinflammation plays an important role in the pathological cascade of Alzheimer's disease (AD) along with aggregation of extracellular amyloid-β (Aβ) plaques and intracellular aggregates of tau protein. In animal models of amyloidosis, local immune activation is centered around Aβ plaques, which are usually of uniform morphology, dependent on the transgenic model used. In postmortem human brains a diversity of Aβ plaque morphologies is seen including diffuse plaques (non-neuritic plaques, non-NP), dense-core plaques, cotton-wool plaques, and NP. In a recent study, we demonstrated that during the progression of Alzheimer's disease neuropathologic changes (ADNC), a transformation of non-NP into NP occurs which is tightly linked to the emergence of cortical, but not hippocampal neurofibrillary tangle (NFT) pathology. This highlights the central role of NP in AD pathogenesis as well as brain region-specific differences in NP formation. In order to correlate the transformation of plaque types with local immune activation, we quantified the clustering and phenotype of microglia and accumulation of astrocytes around non-NP and NP during the progression of ADNC. We hypothesize that glial clustering occurs in response to formation of neuritic dystrophy around NP. First, we show that Iba1-positive microglia preferentially cluster around NP. Utilizing microglia phenotypic markers, we furthermore demonstrate that CD68-positive phagocytic microglia show a strong preference to cluster around NP in both the hippocampus and frontal cortex. A similar preferential clustering is observed for CD11c and ferritin-positive microglia in the frontal cortex, while this preference is less pronounced in the hippocampus, highlighting differences between hippocampal and cortical Aβ plaques. Glial fibrillary acidic protein-positive astrocytes showed a clear preference for clustering around NP in both the frontal cortex and hippocampus. These data support the notion that NP are intimately associated with the neuroimmune response in AD and underscore the importance of the interplay of protein deposits and the immune system in the pathophysiology of AD.