Digestion of whey peptide induces antioxidant and anti-inflammatory bioactivity on glial cells: Sequences identification and structural activity analysis.

Whey derived peptides have shown potential activity improving brain function in pathological condition. However, there is little information about their mechanism of action on glial cells, which have important immune functions in brain. Astrocytes and microglia are essential in inflammatory and oxidative defense that take place in neurodegenerative disease. In this work we evaluate antioxidant and anti-inflammatory potential bioactivity of whey peptide in glial cells. Peptides were formed during simulated gastrointestinal digestion (Infogest protocol), and low molecular weight (<5kDA) peptides (WPHf) attenuated reactive oxygen species (ROS) production induced by hydrogen peroxide stimulus in both cells in dose-dependent manner. WPHf induced an increase in the antioxidant glutathione (GSH) content and prevented GSH reduction induced by lipopolysaccharides (LPS) stimulus in astrocytes cells in a cell specific form. An increase in cytokine mRNA expression (TNFα and IL6) and nitric oxide secretion induced by LPS was attenuated by WPHf pre-treatment in both cells. The inflammatory pathway was dependent on NFκB activation. Bioactive peptide ranking analysis showed positive correlation with hydrophobicity and negative correlation with high molecular weights. The sequence identification revealed 19 peptides cross-referred with bioactive database. Whey peptides were rich in leucine, valine and tyrosine in the C-terminal region and lysine in the N-terminal region. The anti-inflammatory and antioxidant potential of whey peptides were assessed in glia cells and its mechanisms of action were related, such as modulation of antioxidant enzymes and anti-inflammatory pathways. Features of the peptide structure, such as molecular size, hydrophobicity and types of amino acids present in the terminal region are associated to bioactivity.

Biomarker Identification for Gender Specificity of Alzheimer's Disease Based on the Glial Transcriptome Profiles.

Many sex-specific biomarkers have been recently revealed in Alzheimer's disease (AD); however, cerebral glial cells were rarely reported. This study analyzed 220,095 single-nuclei transcriptomes from the frontal cortex of thirty-three AD individuals in the GEO database. Sex-specific Differentially Expressed Genes (DEGs) were identified in glial cells, including 243 in astrocytes, 1,154 in microglia, and 572 in oligodendrocytes. Gene Ontology (GO) functional annotation analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed functional concentration in synaptic, neural, and hormone-related pathways. Protein-protein interaction network (PPI) identified MT3, CALM2, DLG2, KCND2, PAKACB, CAMK2D, and NLGN4Y in astrocytes, TREM2, FOS, APOE, APP, and NLGN4Y in microglia, and GRIN2A, ITPR2, GNAS, and NLGN4Y in oligodendrocytes as key genes. NLGN4Y was the only gene shared by the three glia and was identified as the biomarker for the gender specificity of AD. Gene-transcription factor (TF)-miRNA coregulatory network identified key regulators for NLGN4Y and its target TCMs. Ecklonia kurome Okam (Kunbu) and Herba Ephedrae (Mahuang) were identified, and the effects of the active ingredients on AD were displayed. Finally, enrichment analysis of Kunbu and Mahuang suggested that they might act as therapeutic candidates for gender specificity of AD.

Decoding transcriptomic signatures of cysteine string protein alpha-mediated synapse maintenance.

Synapse maintenance is essential for generating functional circuitry, and decrement in this process is a hallmark of neurodegenerative disease. Yet, little is known about synapse maintenance in vivo. Cysteine string protein α (CSPα), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single-nucleus transcriptomics on the cortex of young CSPα knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in the CSPα KO brain. Significantly, all neuronal classes in CSPα KO brains show strong signatures of repression in synaptic pathways, while up-regulating autophagy-related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. Glial responses varied by cell type, with microglia exhibiting activation. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSPα KO mice. This was mediated by synaptogenic adhesion molecules, with the classical Neurexin1-Neuroligin 1 pair being the most prominent, suggesting that communication of glial cells with neurons is strengthened in CSPα KO mice to preserve synapse maintenance. Together, this study provides a rich dataset of transcriptional changes in the CSPα KO cortex and reveals insights into synapse maintenance and neurodegeneration.

ESCRT disruption provides evidence against trans-synaptic signaling via extracellular vesicles.

Extracellular vesicles (EVs) are released by many cell types, including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating endosomal sorting complex required for transport (ESCRT) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo evenness interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell-autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.

Co-aggregation with Apolipoprotein E modulates the function of Amyloid-ß in Alzheimer's disease.

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer's Disease (AD), but the mechanism underlying this link is poorly understood. In particular, the relevance of direct interactions between apoE and amyloid-ß (Aß) remains controversial. Here, single-molecule imaging shows that all isoforms of apoE associate with Aß in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aß co-aggregates account for ~50% of the mass of diffusible Aß aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aß tune disease-related functions of Aß aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how, in an isoform- and lipidation-specific way, apoE modulates the aggregation, clearance and toxicity of Aß. Selectively removing non-lipidated apoE4-Aß co-aggregates enhances clearance of toxic Aß by glial cells, and reduces secretion of inflammatory markers and membrane damage, demonstrating a clear path to AD therapeutics.

Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

Functional implications of NHR-210 enrichment in C. elegans cephalic sheath glia: insights into metabolic and mitochondrial disruptions in Parkinson's disease models.

Glial cells constitute nearly half of the mammalian nervous system's cellular composition. The glia in C. elegans perform majority of tasks comparable to those conducted by their mammalian equivalents. The cephalic sheath (CEPsh) glia, which are known to be the counterparts of mammalian astrocytes, are enriched with two nuclear hormone receptors (NHRs)-NHR-210 and NHR-231. This unique enrichment makes the CEPsh glia and these NHRs intriguing subjects of study concerning neuronal health. We endeavored to assess the role of these NHRs in neurodegenerative diseases and related functional processes, using transgenic C. elegans expressing human alpha-synuclein. We employed RNAi-mediated silencing, followed by behavioural, functional, and metabolic profiling in relation to suppression of NHR-210 and 231. Our findings revealed that depleting nhr-210 changes dopamine-associated behaviour and mitochondrial function in human alpha synuclein-expressing strains NL5901 and UA44, through a putative target, pgp-9, a transmembrane transporter. Considering the alteration in mitochondrial function and the involvement of a transmembrane transporter, we performed metabolomics study via HR-MAS NMR spectroscopy. Remarkably, substantial modifications in ATP, betaine, lactate, and glycine levels were seen upon the absence of nhr-210. We also detected considerable changes in metabolic pathways such as phenylalanine, tyrosine, and tryptophan biosynthesis metabolism; glycine, serine, and threonine metabolism; as well as glyoxalate and dicarboxylate metabolism. In conclusion, the deficiency of the nuclear hormone receptor nhr-210 in alpha-synuclein expressing strain of C. elegans, results in altered mitochondrial function, coupled with alterations in vital metabolite levels. These findings underline the functional and physiological importance of nhr-210 enrichment in CEPsh glia.

Human-specific insights into candidate genes and boosted discoveries of novel loci illuminate roles of neuroglia in reading disorders.

Reading disorders (RD) are human-specific neuropsychological conditions associated with decoding printed words and/or reading comprehension. So far only a handful of candidate genes segregated in families and 42 loci from genome-wide association study (GWAS) have been identified that jointly provided little clues of pathophysiology. Leveraging human-specific genomic information, we critically assessed the RD candidates for the first time and found substantial human-specific features within. The GWAS candidates (i.e., population signals) were distinct from the familial counterparts and were more likely pleiotropic in neuropsychiatric traits and to harbor human-specific regulatory elements (HSREs). Candidate genes associated with human cortical morphology indeed showed human-specific expression in adult brain cortices, particularly in neuroglia likely regulated by HSREs. Expression levels of candidate genes across human brain developmental stages showed a clear pattern of uplifted expression in early brain development crucial to RD development. Following the new insights and loci pleiotropic in cognitive traits, we identified four novel genes from the GWAS sub-significant associations (i.e., FOXO3, MAPT, KMT2E and HTT) and the Semaphorin gene family with functional priors (i.e., SEMA3A, SEMA3E and SEMA5B). These novel genes were related to neuronal plasticity and disorders, mostly conserved the pattern of uplifted expression in early brain development and had evident expression in cortical neuroglial cells. Our findings jointly illuminated the association of RD with neuroglia regulation-an emerging hotspot in studying neurodevelopmental disorders, and highlighted the need of improving RD phenotyping to avoid jeopardizing future genetic studies of RD.

The Role of Glial Cells in Neurobiology and Prion Neuropathology.

Prion diseases are rare and neurodegenerative diseases that are characterized by the misfolding and infectious spread of the prion protein in the brain, causing progressive and irreversible neuronal loss and associated clinical and behavioral manifestations in humans and animals, ultimately leading to death. The brain has a complex network of neurons and glial cells whose crosstalk is critical for function and homeostasis. Although it is established that prion infection of neurons is necessary for clinical disease to occur, debate remains in the field as to the role played by glial cells, namely astrocytes and microglia, and whether these cells are beneficial to the host or further accelerate disease. Here, we review the current literature assessing the complex morphologies of astrocytes and microglia, and the crosstalk between these two cell types, in the prion-infected brain.

Modification of Gas6 Protein in the Brain by a Functional Endogenous Tissue Vitamin K Cycle.

The TAM receptor ligand Gas6 is known for regulating inflammatory and immune pathways in various organs including the brain. Gas6 becomes fully functional through the post-translational modification of multiple glutamic acid residues into γ-carboxyglutamic in a vitamin K-dependent manner. However, the significance of this mechanism in the brain is not known. We report here the endogenous expression of multiple components of the vitamin K cycle within the mouse brain at various ages as well as in distinct brain glial cells. The brain expression of all genes was increased in the postnatal ages, mirroring their profiles in the liver. In microglia, the proinflammatory agent lipopolysaccharide caused the downregulation of all key vitamin K cycle genes. A secreted Gas6 protein was detected in the medium of both mouse cerebellar slices and brain glial cell cultures. Furthermore, the endogenous Gas6 γ-carboxylation level was abolished through incubation with the vitamin K antagonist warfarin and could be restored through co-incubation with vitamin K1. Finally, the γ-carboxylation level of the Gas6 protein within the brains of warfarin-treated rats was found to be significantly reduced ex vivo compared to the control brains. In conclusion, we demonstrated for the first time the existence of a functional vitamin K cycle within rodent brains, which regulates the functional modification of endogenous brain Gas6. These results indicate that vitamin K is an important nutrient for the brain. Furthermore, the measurement of vitamin K-dependent Gas6 functionality could be an indicator of homeostatic or disease mechanisms in the brain, such as in neurological disorders where Gas6/TAM signalling is impaired.

A Proteomic Approach Identified TFEB as a Key Player in the Protective Action of Novel CB2R Bitopic Ligand FD22a against the Deleterious Effects Induced by ß-Amyloid in Glial Cells.

Neurodegenerative diseases (NDDs) are progressive multifactorial disorders of the nervous system sharing common pathogenic features, including intracellular misfolded protein aggregation, mitochondrial deficit, and inflammation. Taking into consideration the multifaceted nature of NDDs, development of multitarget-directed ligands (MTDLs) has evolved as an attractive therapeutic strategy. Compounds that target the cannabinoid receptor type II (CB2R) are rapidly emerging as novel effective MTDLs against common NDDs, such as Alzheimer's disease (AD). We recently developed the first CB2R bitopic/dualsteric ligand, namely FD22a, which revealed the ability to induce neuroprotection with fewer side effects. To explore the potential of FD22a as a multitarget drug for the treatment of NDDs, we investigated here its ability to prevent the toxic effect of ß-amyloid (Aß25-35 peptide) on human cellular models of neurodegeneration, such as microglia (HMC3) and glioblastoma (U87-MG) cell lines. Our results displayed that FD22a efficiently prevented Aß25-35 cytotoxic and proinflammatory effects in both cell lines and counteracted ß-amyloid-induced depression of autophagy in U87-MG cells. Notably, a quantitative proteomic analysis of U87-MG cells revealed that FD22a was able to potently stimulate the autophagy-lysosomal pathway (ALP) by activating its master transcriptional regulator TFEB, ultimately increasing the potential of this novel CB2R bitopic/dualsteric ligand as a multitarget drug for the treatment of NDDs.

Glia in inhibitory synaptogenesis.

Astrocyte-secreted neurocan guides the formation of inhibitory circuits in the brain.

Single-nucleus analysis reveals microenvironment-specific neuron and glial cell enrichment in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complicated neurodegenerative disease. Neuron-glial cell interactions are an important but not fully understood process in the progression of AD. We used bioinformatic methods to analyze single-nucleus RNA sequencing (snRNA-seq) data to investigate the cellular and molecular biological processes of AD. METHOD: snRNA-seq data were downloaded from Gene Expression Omnibus (GEO) datasets and reprocessed to identify 240,804 single nuclei from healthy controls and patients with AD. The cellular composition of AD was further explored using Uniform Manifold Approximation and Projection (UMAP). Enrichment analysis for the functions of the DEGs was conducted and cell development trajectory analyses were used to reveal underlying cell fate decisions. iTALK was performed to identify ligand-receptor pairs among various cell types in the pathological ecological microenvironment of AD. RESULTS: Six cell types and multiple subclusters were identified based on the snRNA-seq data. A subcluster of neuron and glial cells co-expressing lncRNA-SNHG14, myocardin-related transcription factor A (MRTFA), and MRTFB was found to be more abundant in the AD group. This subcluster was enriched in mitogen-activated protein kinase (MAPK)-, immune-, and apoptosis-related pathways. Through molecular docking, we found that lncRNA-SNHG14 may bind MRTFA and MRTFB, resulting in an interaction between neurons and glial cells. CONCLUSIONS: The findings of this study describe a regulatory relationship between lncRNA-SNHG14, MRTFA, and MRTFB in the six main cell types of AD. This relationship may contribute to microenvironment remodeling in AD and provide a theoretical basis for a more in-depth analysis of AD.

Plp1-expresssing perineuronal DRG cells facilitate colonic and somatic chronic mechanical pain involving Piezo2 upregulation in DRG neurons.

Satellite glial cells (SGCs) of dorsal root ganglia (DRGs) are activated in a variety of chronic pain conditions; however, their mediation roles in pain remain elusive. Here, we take advantage of proteolipid protein (PLP)/creERT-driven recombination in the periphery mainly occurring in SGCs of DRGs to assess the role of SGCs in the regulation of chronic mechanical hypersensitivity and pain-like responses in two organs, the distal colon and hindpaw, to test generality. We show that PLP/creERT-driven hM3Dq activation increases, and PLP/creERT-driven TrkB.T1 deletion attenuates, colon and hindpaw chronic mechanical hypersensitivity, positively associating with calcitonin gene-related peptide (CGRP) expression in DRGs and phospho-cAMP response element-binding protein (CREB) expression in the dorsal horn of the spinal cord. Activation of Plp1+ DRG cells also increases the number of small DRG neurons expressing Piezo2 and acquiring mechanosensitivity and leads to peripheral organ neurogenic inflammation. These findings unravel a role and mechanism of Plp1+ cells, mainly SGCs, in the facilitation of chronic mechanical pain and suggest therapeutic targets for pain mitigation.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Emerging Role of ABC Transporters in Glia Cells in Health and Diseases of the Central Nervous System.

ATP-binding cassette (ABC) transporters play a crucial role for the efflux of a wide range of substrates across different cellular membranes. In the central nervous system (CNS), ABC transporters have recently gathered significant attention due to their pivotal involvement in brain physiology and neurodegenerative disorders, such as Alzheimer's disease (AD). Glial cells are fundamental for normal CNS function and engage with several ABC transporters in different ways. Here, we specifically highlight ABC transporters involved in the maintenance of brain homeostasis and their implications in its metabolic regulation. We also show new aspects related to ABC transporter function found in less recognized diseases, such as Huntington's disease (HD) and experimental autoimmune encephalomyelitis (EAE), as a model for multiple sclerosis (MS). Understanding both their impact on the physiological regulation of the CNS and their roles in brain diseases holds promise for uncovering new therapeutic options. Further investigations and preclinical studies are warranted to elucidate the complex interplay between glial ABC transporters and physiological brain functions, potentially leading to effective therapeutic interventions also for rare CNS disorders.

Uncertainty quantification and sensitivity analysis of neuron models with ion concentration dynamics.

This paper provides a comprehensive and computationally efficient case study for uncertainty quantification (UQ) and global sensitivity analysis (GSA) in a neuron model incorporating ion concentration dynamics. We address how challenges with UQ and GSA in this context can be approached and solved, including challenges related to computational cost, parameters affecting the system's resting state, and the presence of both fast and slow dynamics. Specifically, we analyze the electrodiffusive neuron-extracellular-glia (edNEG) model, which captures electrical potentials, ion concentrations (Na+, K+, Ca2+, and Cl-), and volume changes across six compartments. Our methodology includes a UQ procedure assessing the model's reliability and susceptibility to input uncertainty and a variance-based GSA identifying the most influential input parameters. To mitigate computational costs, we employ surrogate modeling techniques, optimized using efficient numerical integration methods. We propose a strategy for isolating parameters affecting the resting state and analyze the edNEG model dynamics under both physiological and pathological conditions. The influence of uncertain parameters on model outputs, particularly during spiking dynamics, is systematically explored. Rapid dynamics of membrane potentials necessitate a focus on informative spiking features, while slower variations in ion concentrations allow a meaningful study at each time point. Our study offers valuable guidelines for future UQ and GSA investigations on neuron models with ion concentration dynamics, contributing to the broader application of such models in computational neuroscience.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Single-cell multi-cohort dissection of the schizophrenia transcriptome.

The complexity and heterogeneity of schizophrenia have hindered mechanistic elucidation and the development of more effective therapies. Here, we performed single-cell dissection of schizophrenia-associated transcriptomic changes in the human prefrontal cortex across 140 individuals in two independent cohorts. Excitatory neurons were the most affected cell group, with transcriptional changes converging on neurodevelopment and synapse-related molecular pathways. Transcriptional alterations included known genetic risk factors, suggesting convergence of rare and common genomic variants on neuronal population-specific alterations in schizophrenia. Based on the magnitude of schizophrenia-associated transcriptional change, we identified two populations of individuals with schizophrenia marked by expression of specific excitatory and inhibitory neuronal cell states. This single-cell atlas links transcriptomic changes to etiological genetic risk factors, contextualizing established knowledge within the human cortical cytoarchitecture and facilitating mechanistic understanding of schizophrenia pathophysiology and heterogeneity.