Cellular and Molecular Neurobiology

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Microglial cells constantly surveil the cerebral microenvironment and become activated following injury and disease to mediate inflammatory responses. The nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome, which is abundantly expressed in microglial cells, plays a key role in these responses as well as in the development of many neurological disorders. Microglial cell lines are a valuable tool to study the causes and possible treatments for neurological diseases which are linked to inflammation. Here, we investigated whether the mouse microglial cell line IMG is suitable to study NLRP3 inflammasome by incubating cells with different concentrations of NLRP3 inflammasome priming and activating agents lipopolysaccharide (LPS) and ATP, respectively, and applying short (4 h) or long (24 h) LPS incubation times. After short LPS incubation, the mRNA levels of most pro-inflammatory and NLRP3 inflammasome-associated genes were more upregulated than after long incubation. Moreover, the combination of higher LPS and ATP concentrations with short incubation time resulted in greater levels of active forms of caspase-1 and interleukin-1 beta (IL-1β) proteins than low LPS and ATP concentrations or long incubation time. We also demonstrated that treatment with NLRP3 inflammasome inhibitor glibenclamide suppressed NLRP3 inflammasome activation in IMG cells, as illustrated by the downregulation of gasdermin D N-fragment and mature caspase-1 and IL-1β protein levels. In addition, we conducted similar experiments with primary microglial cells and BV-2 cell line to determine the similarities and differences in their responses. Overall, our results indicate that IMG cell line could be a valuable tool for NLRP3 inflammasome studies. Graphical Abstract In IMG cells, 4-h incubation with lipopolysaccharide (LPS) induces a stronger upregulation of NLRP3 inflammasome-associated pro-inflammatory genes compared to 24-h incubation. NLRP3 inflammasome is robustly activated only after the addition of 3 mM of ATP following short LPS incubation time.
 
A significant amount of evidence from the past few years has shown that Sirtuin 1 (SIRT1), a histone deacetylase dinucleotide of nicotinamide adenine dinucleotide (NAD+) is closely related to the cerebral ischemia. Several potential neuroprotective strategies like resveratrol, ischemia preconditioning, and caloric restriction exert their neuroprotection effects through SIRT1-related signaling pathway. However, the potential mechanisms and neuroprotection of SIRT1 in the process of cerebral ischemia injury development and recovery have not been systematically elaborated. This review summarized the the deacetylase activity and distribution of SIRT1 as well as analyzed the roles of SIRT1 in astrocytes, microglia, neurons, and brain microvascular endothelial cells (BMECs), and the molecular mechanisms of SIRT1 in cerebral ischemia, providing a theoretical basis for exploration of new therapeutic target in future.
 
Chronic diabetic conditions have been associated with certain cerebral complications, that include neurobehavioral dysfunctional patterns and morphological alterations of neurons, especially the hippocampus. Neuroanatomical studies done by the authors have shown decreased total dendritic length, intersections, dendritic length per branch order and nodes in the CA1 hippocampal region of the diabetic brain as compared to its normal control group, indicating reduced dendritic arborization of the hippocampal CA1 neurons. Epigenetic alterations in the brain are well known to affect age-associated disorders, however its association with the evolving diabetes-induced damage in the brain is still not fully understood. DNA hypermethylation within the neurons, tend to silent the gene expression of several regulatory proteins. The findings in the study have shown an increase in global DNA methylation in palmitic acid-induced lipotoxic Neuro-2a cells as well as within the diabetic mice brain. Inhibiting DNA methylation, restored the levels of HSF1 and certain HSPs, suggesting plausible effect of DNMTs in maintaining the proteostasis and synaptic fidelity. Neuroinflammation, as exhibited by the astrocyte activation (GFAP), were further significantly decreased in the 5-azadeoxycytidine group (DNMT inhibitor). This was further evidenced by decrease in proinflammatory cytokines TNF⍺, IL-6, and mediators iNOS and Phospho-NFkB. Our results suggest that changes in DNA methylation advocate epigenetic dysregulation and its involvement in disrupting the synaptic exactitude in the hippocampus of diabetic mice model, providing an insight into the pathophysiology of diabetes-induced neuroepigenetic changes.
 
Spinal cord injury (SCI) leads to long-term and permanent motor dysfunctions, and nervous system abnormalities. Injury to the spinal cord triggers a signaling cascade that results in activation of the inflammatory cascade, apoptosis, and Zn(II) ion homeostasis. Trehalose (Tre), a nonreducing disaccharide, and l-carnosine (Car), (β-alanyl-l-histidine), one of the endogenous histidine dipeptides have been recognized to suppress early inflammatory effects, oxidative stress and to possess neuroprotective effects. We report on the effects of the conjugation of Tre with Car (Tre–car) in reducing inflammation in in vitro and in vivo models. The in vitro study was performed using rat pheochromocytoma cells (PC12 cell line). After 24 h, Tre–car, Car, Tre, and Tre + Car mixture treatments, cells were collected and used to investigate Zn²⁺ homeostasis. The in vivo model of SCI was induced by extradural compression of the spinal cord at the T6–T8 levels. After treatments with Tre, Car and Tre–Car conjugate 1 and 6 h after SCI, spinal cord tissue was collected for analysis. In vitro results demonstrated the ionophore effect and chelating features of l-carnosine and its conjugate. In vivo, the Tre–car conjugate treatment counteracted the activation of the early inflammatory cascade, oxidative stress and apoptosis after SCI. The Tre–car conjugate stimulated neurotrophic factors release, and influenced Zn²⁺ homeostasis. We demonstrated that Tre–car, Tre and Car treatments improved tissue recovery after SCI. Tre–car decreased proinflammatory, oxidative stress mediators release, upregulated neurotrophic factors and restored Zn²⁺ homeostasis, suggesting that Tre–car may represent a promising therapeutic agent for counteracting the consequences of SCI.
 
Substance-P (SP) is an 11 amino acid neuropeptide that is known to stimulate the peripheral mobilization of bone marrow mesenchymal stem cells and M2 polarization in monocytes/macrophages in a variety of acute and chronic tissue injuries. To examine the role of SP in protection and recovery from acute ischemic brain injury, experimental ischemic stroke was induced by transient middle cerebral artery occlusion (tMCAo) in rats for 1 h with subsequent reperfusion. Two injections of SP, immediately and one day post-tMCAo, resulted in approximately threefold lower mortality and 40% less infarct volume than those of saline-treated rats at seven days post-tMCAo. At 4.5 h, SP markedly increased CD11b/c⁺CD163⁺/CD 206⁺ cells in the blood, which were concomitantly decreased in the bone marrow, suggesting that SP preferentially mobilized M2-polarized monocytes. After two days, SP increased the expression of neuroprotective and anti-inflammatory genes in the ischemic brain and induced neuronal survival in the brain penumbra. Additionally, SP markedly increased CD68⁺CD163⁺ and CD68⁺CD206⁺ M2 microglia/macrophages in the ischemic brain during seven days post-tMCAo. Furthermore, SP preserved the blood‒brain barrier in the ischemic brain, which was confirmed by the abundant levels of SMI71⁺ brain endothelial cells that colocalized with α-SMA⁺ pericytes. The beneficial effects of SP on functional recovery and tissue preservation were maintained for six weeks. Collectively, SP treatment in the early phase of ischemic stroke markedly suppressed the destructive inflammatory response and improved the microenvironment for tissue protection and repair.
 
The interaction and regulation network between m6A modification regulators and genes/pathways.
Gliomas are the most common central cancer with high aggressive-capacity and poor prognosis, remaining to be the threat of most patients. With the blood–brain barrier and highly malignant progression, the efficacy of high-intensity treatment is limited. The N6-methyladenine (m⁶A) modification is found in rRNA, snRNA, miRNA, lncRNA, and mRNA, influencing the metabolism and translation of these RNAs and consequently regulating the proliferation, metastasis, apoptosis, etc. of glioma cells. The key role that m⁶A modification in gliomas has played makes it a prospective target for diagnosis and treatment. However, with studying deeper in m⁶A modification and gliomas, the conclusion and mechanism are abundant and complex. This review focused on the dysregulation of m⁶A regulators and m⁶A modification of key genes and pathways in Hallmarks of gliomas. Furthermore, the potential of exploiting m⁶A modification for gliomas diagnosis and therapeutics was also discussed. This review will summarize the recent studies about m⁶A modification, revealing that m⁶A modification plays an important role in the malignant progression, angiogenesis, microenvironment, and genome instability in gliomas by exploring the interaction and network between m⁶A modification-related regulators and classical tumor-related genes. And it might provide some clue for the molecular mechanism, diagnosis, and treatment of gliomas. Graphical Abstract
 
Long-COVID is a pathology interesting multiple organs. Gastrointestinal and neurological disorders, loss of body weight, and muscle mass can be observed. The symptoms also extend to the psychological sphere. Kidneys and cardiocirculatory disorders due to long-COVID-19 are not treated in the present review. Image created with BioRender.com
Intervention plan for long-COVID home management. Image created with BioRender.com
SARS-CoV-2 pandemic has caused a collapse of the world health systems. Now, vaccines and more effective therapies have reversed this crisis but the scenario is further aggravated by the appearance of a new pathology, occurring as SARS-CoV-2 infection consequence: the long-COVID-19. This term is commonly used to describe signs and symptoms that continue or develop after acute infection of COVID-19 up to several months. In this review, the consequences of the disease on mental health and the neurological implications due to the long-COVID are described. Furthermore, the appropriate nutritional approach and some recommendations to relieve the symptoms of the pathology are presented. Data collected indicated that in the next future the disease will affect an increasing number of individuals and that interdisciplinary action is needed to counteract it.
 
The success rate of regenerative medicine largely depends on the type of stem cells applied in such procedures. Consequently, to achieve the needed level for clinical standardization, we need to investigate the viability of accessible sources with sufficient quantity of cells. Since the oral region partly originates from the neural crest, which naturally develops in niche with decreased levels of oxygen, the main goal of this work was to test if human oral mucosa stem cells (hOMSC) might be used to treat neurons damaged by anoxia. Here we show that hOMSC are more resistant to anoxia than human induced pluripotent stem cells and that they secrete BDNF, GDNF, VEGF and NGF. When hOMSC were added to human neurons damaged by anoxia, they significantly improved their survival. This regenerative capability was at least partly achieved through miR-514A-3p and SHP-2 and it decreased in hOMSC exposed to neural cells for 14 or 28 days. In addition, the beneficial effect of hOMSC were also confirmed in mice affected by stroke. Hence, in this work we have confirmed that hOMSC, in a time-limited manner, improve the survival of anoxia-damaged neurons and significantly contribute to the recovery of experimental animals following stroke.
 
To investigate the characteristics of mutation myocilin proteins and glaucoma pathological phenotype in transgenic mice with full-length human Pro370Leu mutant myocilin gene (Tg-MYOCP370L). Tg-MYOCP370L mice were established using the CRISPR/Cas9 system. Long-term intraocular pressure (IOP) was measured, myocilin protein expressions in anterior chamber angle, retina, optic nerve tissues and aqueous humor were detected by western blot. RBPMS, myocilin, Iba-1 and GFAP expression were visualized by immunofluorescence. H&E staining was applied to assess the ocular angle and retinal morphology. Aqueous humor dynamics were visualized by Gadolinium magnetic resonance imaging (Gd-MRI). TUNEL assay was used to evaluate the specific cell apoptosis in trabecular meshwork and retina. Optomotor and electroretinography tests were employed to evaluate the visual function in Tg-MYOCP370L and wild-type (WT) mice. Homozygous myocilin mutation at position 503 (C > T) was identified by PCR and sequencing in Tg-MYOCP370L mice. Myocilin protein expression was overexpressed in eye tissues of Tg-MYOCP370L mice with reduced myocilin secretion in aqueous humor. H&E staining showed normal histological morphology of anterior chamber angle whereas decreased thickness and nuclei in ganglion cell layer were found (P < 0.05). Gd signals were significantly increased in the anterior chamber of Tg-MYOCP370L compared with WT eyes (P < 0.05). IOP was elevated in Tg-MYOCP370L mice starting at 5 months of age, with significant RGC loss (P < 0.05). Upregulation of caspase-3 and caspase-9 expressions and increased TUNEL-positive cells were found in eyes of Tg-MYOCP370L mice. Excessive activation of retinal glial cells and impaired visual function were detected in Tg-MYOCP370L mice. Tg-MYOCP370L mice can induce the phenotype of open-angle glaucoma, featured as IOP elevation, activated retinal glial cells, loss of RGCs and impaired visual function. These pathologic changes may arise from the abnormal mutant myocilin protein accumulation in the trabecular meshwork and injured aqueous humor drainage. Therefore, Tg-MYOCP370L mice model can serve as an effective animal model for glaucoma research, especially for glaucoma-associated myocilin mutation studies.
 
Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aβ 1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aβ 1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aβ 1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD. Graphical Abstract Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.
 
The feeding-related hormone, acyl-ghrelin, protects dopamine neurones in murine 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-based models of experimental Parkinson’s disease (PD). However, the potential protective effect of acyl-ghrelin on substantia nigra pars compacta (SNpc) dopaminergic neurones and consequent behavioural correlates in the more widely used 6-hydroxydopamine (6-OHDA) rat medial forebrain bundle (MFB) lesion model of PD are unknown. To address this question, acyl-ghrelin levels were raised directly by mini-pump infusion for 7 days prior to unilateral injection of 6-OHDA into the MFB with assessment of amphetamine-induced rotations on days 27 and 35, and immunohistochemical analysis of dopaminergic neurone survival. Whilst acyl-ghrelin treatment was insufficient to elevate food intake or body weight, it attenuated amphetamine-induced circling behaviour and SNpc dopamine neurone loss induced by 6-OHDA. These data support the notion that elevating circulating acyl-ghrelin may be a valuable approach to slow or impair progression of neurone loss in PD. Graphical abstract
 
Acute ischemic stroke (AIS) is a serious threat to human health. Following AIS, cerebral ischemia–reperfusion injury (CIRI) must be treated to improve prognosis. By combining 4D label-free quantitative proteomics with lactylation modification-specific proteomics analysis, we assessed lysine lactylation (Kla) in cortical proteins of a CIRI rat model. We identified a total of 1003 lactylation sites on 469 proteins in this study, gathering quantitative information (PXD034232) on 660 of 310 proteins, which were further classified by cell composition, molecular function, and biological processes. In addition, we analyzed the metabolic pathways, domains, and protein–protein interaction networks. Lastly, we evaluated differentially expressed lysine lactylation sites, determining 49 upregulated proteins and 99 downregulated proteins with 54 upregulated sites and 54 downregulated sites in the experimental group in comparison with the healthy control group. Moreover, we identified the Kla of Scl25a4 and Slc25a5 in the Ca²⁺ signaling pathway, but the Kla of Vdac1 was eliminated, as confirmed in vivo. Overall, these results provide new insights into lactylation involved in the underlying mechanism of CIRI because this post-translational modification affects the mitochondrial apoptosis pathway and mediates neuronal death. Therefore, this study may enable us to develop new molecules with therapeutic properties, which have both theoretical significance and broad clinical application prospects. Graphical Abstract A new model of cerebral ischemia–reperfusion injury (CIRI) induced by lactylation through the regulation of key proteins of the Ca²⁺ signaling pathway.
 
After restoration of spontaneous circulation (ROSC) following cardiac arrest, complements can be activated and excessive autophagy can contribute to the brain ischemia–reperfusion (I/R) injury. Mild hypothermia (HT) protects against brain I/R injury after ROSC, but the mechanisms have not been fully elucidated. Here, we found that HT significantly inhibited the increases in serum NSE, S100β, and C5a, as well as neurologic deficit scores, TUNEL-positive cells, and autophagic vacuoles in the pig brain cortex after ROSC. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen–glucose deprivation/reoxygenation. HT could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Our findings demonstrated that C5a could bind to C5aR1 to induce neuronal autophagy during the brain I/R injury, which was associated with the inhibited PI3K/Akt/mTOR pathway. HT could inhibit C5a-induced neuronal autophagy by regulating the C5a–C5aR1 interaction and the PI3K/Akt/mTOR pathway, which might be one of the neuroprotective mechanisms underlying I/R injury. Graphical Abstract The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen–glucose deprivation/reoxygenation. Mild hypothermia (HT) could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Proposed mechanism by which HT protects against brain I/R injury by repressing C5a–C5aR1-induced excessive autophagy. Complement activation in response to brain I/R injury generates C5a that can interact with C5aR1 to inactivate mTOR, probably through the PI3K-AKT pathway, which can finally lead to autophagy activation. The excessively activated autophagy ultimately contributes to cell apoptosis and brain injury. HT may alleviate complement activation and then reduce C5a-induced autophagy to protect against brain I/R injury. HT, mild hypothermia; I/R, ischemia reperfusion.
 
Excessive activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propoinic acid (AMPA) receptors instigates excitotoxicity via enhanced calcium influx in the neurons thus inciting deleterious consequences. Additionally, Endoplasmic Reticulum (ER) is pivotal in maintaining the intracellular calcium balance. Considering this, studying the aftermath of enhanced calcium uptake by neurons and its effect on ER environment can assist in delineating the pathophysiological events incurred by excitotoxicty. The current study was premeditated to decipher the role of ER pertaining to calcium homeostasis in AMPA-induced excitotoxicity. The findings showed, increased intracellular calcium levels (measured by flowcytometry and spectroflourimeter using Fura 2AM) in AMPA excitotoxic animals (male Sprague dawely rats) (intra-hippocampal injection of 10 mM AMPA). Further, ER resident proteins like calnexin, PDI and ERp72 were found to be upregulated, which further modulated the functioning of ER membrane calcium channels viz. IP3R, RyR, and SERCA pump. Altered calcium homeostasis further led to ER stress and deranged the protein folding capacity of ER post AMPA toxicity, which was ascertained by unfolded protein response (UPR) pathway markers such as IRE1α, eIF2α, and ATF6α. Chemical chaperone, 4-phenybutric acid (4-PBA), ameliorated the protein folding capacity and subsequent UPR markers. In addition, modulation of calcium channels and calcium regulating machinery of ER post 4-PBA administration restored the calcium homeostasis. Therefore the study reinforces the significance of ER stress, a debilitating outcome of impaired calcium homeostasis, under AMPA-induced excitotoxicity. Also, employing chaperone-based therapeutic approach to curb ER stress can restore the calcium imbalance in the neuropathological diseases. Graphical Abstract
 
Schematic representation depicting role of exercise responsome on CNS/brain: During physical exercise, body releases different cytokines from different tissues like skeletal muscles (myokines), adipose tissues (adipokines), liver (hepatokines), and bone (osteokines). These cytokines altogether are known as “exerkines.” These exerkines are released in blood and then travel through blood–brain barrier (BBB) and regulate the cellular signaling pathways e.g., IGF-1/PI3K/AKT axis and AMPK/SIRT1/PGC1α axis in CNS/brain causing increased neurogenesis, improved synaptic plasticity, and reduced neuroinflammation. Created with BioRender.com
Exercise-mediated release of various exerkines from different tissues and their effects on hippocampal region of brain: Exercise releases various exerkines like FNDC5/irisin, CTSB, Adiponectin, Osteocalcin, FGF21 from different tissues like skeletal muscles (myokines), adipose tissues (adipokines), liver (hepatokines), and bone (osteokines), respectively. These exerkines cross the BBB and modulate the activities of important signaling molecules such as AKT/ERK, AMPK, cAMP/PKA/CREB, BDNF, and gpr158 which further lead to exercise-induced benefits for brain. Leptin is synthesized and secreted in the brain in response to exercise causing neuroprotection. Created with BioRender.com
Exercise mimetics targeting the AMPK-SIRT1-PGC1α-BDNF signaling pathway to enhance cognitive performance. Exercise mimetics such as AICAR, R419, AdipoRon, GTDF, and GW501516 act on signaling molecules involved in translating exercise-mediated benefits on the brain. They target AMPK, PPARα, PPARẟ, PGC1α, ultimately converging upon BDNF expression. Created with BioRender.com
The beneficial effects of exercise on the proper functioning of the body have been firmly established. Multi-systemic metabolic regulation of exercise is the consequence of multitudinous changes that occur at the cellular level. The exercise responsome comprises all molecular entities including exerkines, miRNA species, growth factors, signaling proteins that are elevated and activated by physical exercise. Exerkines are secretory molecules released by organs such as skeletal muscle, adipose tissue, liver, and gut as a function of acute/chronic exercise. Exerkines such as FNDC5/irisin, Cathepsin B, Adiponectin, and IL-6 circulate through the bloodstream, cross the blood–brain barrier, and modulate the expression of important signaling molecules such as AMPK, SIRT1, PGC1α, BDNF, IGF-1, and VEGF which further contribute to improved energy metabolism, glucose homeostasis, insulin sensitivity, neurogenesis, synaptic plasticity, and overall well-being of the body and brain. These molecules are also responsible for neuroprotective adaptations that exercise confers on the brain and potentially ameliorate neurodegeneration. This review aims to detail important cellular and molecular species that directly or indirectly mediate exercise-induced benefits in the body, with an emphasis on the central nervous system.
 
The main inflammation triggers in Alzheimer’s disease
Interactions between autophagy and inflammation in cellular level
Autophagy is a highly evolutionary conserved process that degrades cytosolic macromolecules or damaged organelles (e.g., mitochondria), as well as intracellular pathogens for energy and survival. Dysfunction of autophagy has been associated with the pathologies of Alzheimer’s disease (AD), including Aβ plaques and neurofibrillary tangles. Recently, the presence of sustained immune response in the brain has been considered a new core pathology in AD. Accumulating evidence suggests that autophagy activation may suppress inflammation response through degrading inflammasomes or pro-inflammatory cytokines and improving immune system function in both clinical trials and preclinical studies. This review provides an overview of updated information on autophagy and inflammation and their potential mediators in AD. In summary, we believe that understanding the relationship between autophagy and inflammation will provide insightful knowledge for future therapeutic implications in AD.
 
Fiber intake is associated with a lower risk for Alzheimer´s disease (AD) in older adults. Intake of plant-based diets rich in soluble fiber promotes the production of short-chain fatty acids (SCFAs: butyrate, acetate, propionate) by gut bacteria. Butyrate administration has antiinflammatory actions, but propionate promotes neuroinflammation. In AD patients, gut microbiota dysbiosis is a common feature even in the prodromal stages of the disease. It is unclear whether the neuroprotective effects of fiber intake rely on gut microbiota modifications and specific actions of SCFAs in brain cells. Here, we show that restoration of the gut microbiota dysbiosis through the intake of soluble fiber resulted in lower propionate and higher butyrate production, reduced astrocyte activation and improved cognitive function in 6-month-old male APP/PS1 mice. The neuroprotective effects were lost in antibiotic-treated mice. Moreover, propionate promoted higher glycolysis and mitochondrial respiration in astrocytes, while butyrate induced a more quiescent metabolism. Therefore, fiber intake neuroprotective action depends on the modulation of butyrate/propionate production by gut bacteria. Our data further support and provide a mechanism to explain the beneficial effects of dietary interventions rich in soluble fiber to prevent dementia and AD. Fiber intake restored the concentration of propionate and butyrate by modulating the composition of gut microbiota in male transgenic (Tg) mice with Alzheimer´s disease. Gut dysbiosis was associated with intestinal damage and high propionate levels in control diet fed-Tg mice. Fiber-rich diet restored intestinal integrity and promoted the abundance of butyrate-producing bacteria. Butyrate concentration was associated with better cognitive performance in fiber-fed Tg mice. A fiber-rich diet may prevent the development of a dysbiotic microbiome and the related cognitive dysfunction in people at risk of developing Alzheimer´s disease.
 
STRING network of the proteins in Table 1. The three adhesion-related biomarkers are positioned at the top; the coagulation-related biomarkers are positioned at the bottom. The immune-inflammation biomarkers are found in the middle of the network (pale red), using the default parameters of the STRING web interface; the layout considers node connectivity. As explained in detail in the online legend associated with the network, there is no particular meaning of the node color. The edge color refers to the source database of the interaction, i.e., curated databases (cyan), experimentally determined (magenta), predicted by gene neighborhood (green), by gene fusions (red) or by gene co-occurrence (blue), or taken from text mining (light green), co-expression (black) or protein homology (light blue) data. The permanent link to the network, including an online legend, is https://version-11-0b.string-db.org/cgi/network?taskId=b4MNWvbtbPcL
Blood-based biomarkers that predict stroke outcomes
Gene ontology biological process enrichment (first 50 terms) provided by STRING for the network of Fig. 1
The most important predictors for outcomes after ischemic stroke, that is, for health deterioration and death, are chronological age and stroke severity; gender, genetics and lifestyle/environmental factors also play a role. Of all these, only the latter can be influenced after the event. Recurrent stroke may be prevented by antiaggregant/anticoagulant therapy, angioplasty of high-grade stenoses, and treatment of cardiovascular risk factors. Blood cell composition and protein biomarkers such as C-reactive protein or interleukins in serum are frequently considered as biomarkers of outcome. Here we aim to provide an up-to-date protein biomarker signature that allows a maximum of mechanistic understanding, to predict health deterioration following stroke. We thus surveyed protein biomarkers that were reported to be predictive for outcome after ischemic stroke, specifically considering biomarkers that predict long-term outcome (≥ 3 months) and that are measured over the first days following the event. We classified the protein biomarkers as immune‑inflammatory, coagulation-related, and adhesion-related biomarkers. Some of these biomarkers are closely related to cellular senescence and, in particular, to the inflammatory processes that can be triggered by senescent cells. Moreover, the processes that underlie inflammation, hypercoagulation and cellular senescence connect stroke to cancer, and biomarkers of cancer-associated thromboembolism, as well as of sarcopenia, overlap strongly with the biomarkers discussed here. Finally, we demonstrate that most of the outcome-predicting protein biomarkers form a close-meshed functional interaction network, suggesting that the outcome after stroke is partially determined by an interplay of molecular processes relating to inflammation, coagulation, cell adhesion and cellular senescence.
 
Schematic presentation of interaction between mitochondrial dysfunction and Parkinson's disease and therapeutic approaches that target mitochondria to treat PD. Intracellular accumulation of α-synuclein, respiratory chain dysfunction, failure in the mechanisms involved in mitophagy processes, and alterations in mtDNA such as depletion, mutation, and deletion are prominent causative factors that link mitochondrial dysfunction to PD. Targeting mitochondria using mitochondrial enhancers, mitophagy enhancers, exercise, and mitochondrial transplantations are among the most important therapeutic approaches that could efficiently implicated for PD treatment
Abstract Neurodegeneration is among the most critical challenges that involve modern societies and annually influences millions of patients worldwide. While the pathophysiology of Parkinson's disease (PD) is complicated, the role of mitochondrial is demonstrated. The in vitro and in vivo models and genome-wide association studies in human cases proved that specific genes, including PINK1, Parkin, DJ-1, SNCA, and LRRK2, linked mitochondrial dysfunction with PD. Also, mitochondrial DNA (mt-DNA) plays an essential role in the pathophysiology of PD. Targeting mitochondria as a therapeutic approach to inhibit or slow down PD formation and progression seems to be an exciting issue. The current review summarized known mutations associated with both mitochondrial dysfunction and PD. Then, the importance of mt-DNA in PD pathogenesis and potential therapeutic approaches for PD targeting mitochondrial dysfunction were discussed. Keywords: Parkinson's disease; mitochondrial dysfunction, neurodegeneration
 
Endothelial cells (ECs) and pericytes are present in all blood vessels. Their position confers an important role in controlling oxygen and nutrient transportation to the different organs. ECs can adopt different morphologies based on their need and functions. Both ECs and pericytes express different surface markers that help in their identification, but heterogeneity and overlapping between markers among different cells pose a challenge for their precise identification. Spatiotemporal association of ECs and pericytes have great importance in sprout formation and vessel stabilization. Any traumatic injury in CNS may lead to vascular damage along with neuronal damage. Hence, ECs–pericyte interaction by physical contact and paracrine molecules is crucial in recovering the epicenter region by promoting angiogenesis. ECs can transform into other types of cells through endothelial–mesenchymal transition (EndMT), promoting wound healing in the epicenter region. Various signaling pathways mediate the interaction of ECs with pericytes that have an extensive role in angiogenesis. In this review, we discussed ECs and pericytes surface markers, the spatiotemporal association and interaction of ECs–pericytes, and signaling associated with the pathology of traumatic SCI. Linking the brain or spinal cord-specific pathologies and human vascular pathology will pave the way toward identifying new therapeutic targets and developing innovative preventive strategies. Graphical Abstract Endothelial-pericyte interaction strategic for formation of functional neo-vessels that are crucial for neurological recovery
 
Oxaliplatin is widely used in cancer treatment, however, many patients will suffer from neuropathic pain (NP) induced by it at the same time. Therefore exploring the mechanism and founding novel target for this problem are needed. In this study, YTHDF1 showed upregulation in oxaliplatin treated mice. As m6A is known as conserved and it widely functions in numerous physiological and pathological processes. Therefore, we focused on exploring the molecular mechanism of whether and how YTHDF1 functions in NP induced by oxaliplatin. IHC and western blotting were conducted to measure proteins. Intrathecal injection for corresponding siRNAs in C57/BL6 mice or spinal microinjection for virus in YTHDF1flox/flox mice were applied to specially knockdown the expression of molecular. Von Frey, acetone test and ethyl chloride (EC) test were applied to evaluate NP behavior. YTHDF1, Wnt3a, TNF-α and IL-18 were increased in oxaliplatin treated mice, restricted the molecular mentioned above respectively can significantly attenuate oxaliplatin-induced NP, including the mechanical allodynia and cold allodynia. Silencing YTHDF1 and inhibiting Wnt3a and Wnt signaling pathways can reduce the enhancement of TNF-α and IL-18, and the decreasing of the upregulation of YTHDF1 can be found when inhibiting Wnt3a and Wnts signaling pathways in oxaliplatin treated mice. Our study indicated a novel pathway that can contribute to oxaliplatin-induced NP, the Wnt3a/YTHDF1 to cytokine pathway, which upregulating YTHDF1 functioned as the downstream of Wnt3a signal and promoted the translation of TNF-α and IL-18 in oxaliplatin treated mice.
 
Human cytomegalovirus (HCMV) causes congenital neurological lifelong disabilities. To date, the neuropathogenesis of brain injury related to congenital HCMV (cCMV) infection is poorly understood. This study evaluates the characteristics and pathogenetic mechanisms of encephalic damage in cCMV infection. Ten HCMV-infected human fetuses at 21 weeks of gestation were examined. Specifically, tissues from different brain areas were analyzed by: (i) immunohistochemistry (IHC) to detect HCMV-infected cell distribution, (ii) hematoxylin–eosin staining to evaluate histological damage and (iii) real-time PCR to quantify tissue viral load (HCMV-DNA). The differentiation stage of HCMV-infected neural/neuronal cells was assessed by double IHC to detect simultaneously HCMV-antigens and neural/neuronal markers: nestin (a marker of neural stem/progenitor cells), doublecortin (DCX, marker of cells committed to the neuronal lineage) and neuronal nuclei (NeuN, identifying mature neurons). HCMV-positive cells and viral DNA were found in the brain of 8/10 (80%) fetuses. For these cases, brain damage was classified as mild (n = 4, 50%), moderate (n = 3, 37.5%) and severe (n = 1, 12.5%) based on presence and frequency of pathological findings (necrosis, microglial nodules, microglial activation, astrocytosis, and vascular changes). The highest median HCMV-DNA level was found in the hippocampus (212 copies/5 ng of human DNA [hDNA], range: 10–7,505) as well as the highest mean HCMV-infected cell value (2.9 cells, range: 0–23), followed by that detected in subventricular zone (1.7 cells, range: 0–19). These findings suggested a preferential viral tropism for both neural stem/progenitor cells and neuronal committed cells, residing in these regions, confirmed by the expression of DCX and nestin in 94% and 63.3% of HCMV-positive cells, respectively. NeuN was not found among HCMV-positive cells and was nearly absent in the brain with severe damage, suggesting HCMV does not infect mature neurons and immature neural/neuronal cells do not differentiate into neurons. This could lead to known structural and functional brain defects from cCMV infection. Graphical Abstract
 
Signaling in the endocannabinoid synapse. Glutamate released by presynaptic cells stimulates ionotropic and metabotropic receptors in postsynaptic cells, leading to an increase in Ca²⁺ influx/concentration. Ca²⁺ and/or Gq stimulate PLC, which leads to the production of DAG, the substrate for DAGL to form 2-AG, while NAPE-PLD hydrolyses NAPE to form AEA. Endocannabinoids diffuse through the synaptic cleft, reaching the CB1 and CB2 receptors TRPV1 and GPR55, among others. The classical downstream pathway of the CB receptor occurs through Gi-protein: inhibition of AC activity, modulation of K⁺ and Ca²⁺ channels, and subsequent inhibition of NT release. The signaling is ended by endocannabinoid degradation. FAAH metabolizes AEA into AA and ethanolamine, while COX-2 can eventually generate prostamide from AEA. MAGL, ABHD12, and ABHD6 degrade 2-AG in AA and glycerol, while COX-2 is metabolized in PGE2-G. The thickness of arrows from metabolizing enzymes represents the importance in the control of AEA and 2-AG availability
Primary metabolic pathways of AEA and 2-AG synthesis and degradation. a. AEA synthesis—An increase in Ca²⁺ stimulates NAPE-PLD to hydrolyze NAPE, producing AEA. PLC catalyzes the formation of phospho-NAE, and the phosphatases PTPN22 and INPP5D then convert phospho-NAE to AEA. NAPE is catalyzed by sPLA2 or ABHD4, generating lyso-NAPE. Then, ABHD4 catalyzes the formation of GP-NAE, which is converted to AEA by the phosphodiesterase GDE1. 2-AG synthesis—PLC mediates the hydrolysis of PIP2, which produces DAG, which is converted to 2-AG by DAGLα/β. Phosphatidic acid (PA) is hydrolyzed by PA phosphohydrolase, producing DAG, which is converted by DAGLα/β into 2-AG. b. AEA degradation—AEA is degraded primarily by FAAH into AA and ethanolamine, while COX-2 degrades into prostamide. 2-AG degradation—2-AG is degraded primarily by MAGL into AA and glycerol. ABHD6 and ABHD12 also degrade 2-AG into AA and glycerol, although they are less common. COX-2 degrades 2-AG into PGE2-G
Apoptosis induced by cannabinoids. AEA and 2-AG activate CB1R and COX-2, inducing apoptosis in a manner dependent on the cholesterol-rich lipid rafts in cultured decidual cells. 2-AG causes an increase in ROS, leading to reticulum stress and apoptosis through PERK-ATF4-CHOP in the placenta via CB2R. 2-AG, Δ9-THC and synthetic cannabinoids increased ROS, causing reticulum stress-induced apoptosis in the cytotrophoblastic cell lineage
Apoptosis induced by cannabinoids in the CNS. CP-55,940 activates CB1R and causes apoptosis via caspase-3 in forebrain cultures. Δ9-THC, through CB1R, leads to cytochrome c release and caspase-3 activation, inducing apoptosis in cultured cortical neuron cells. Δ9-THC increases AA through PLA2, activating COX-2 and culminating in an increase in ROS, causing hippocampal neural death. P2X7 activation increases the activity of NAPE-PLD and DAGL, leading to an increase in 2-AG and AEA. 2-AG, AEA and WIN activate CB1R and CB2R signaling through P2X7R and cytoplasmic Ca²⁺, causing an increase in superoxide and cytochrome c release and leading to apoptosis in the retina during development
The active principles of Cannabis sativa are potential treatments for several diseases, such as pain, seizures and anorexia. With the increase in the use of cannabis for medicinal purposes, a more careful assessment of the possible impacts on embryonic development becomes necessary. Surveys indicate that approximately 3.9% of pregnant women use cannabis in a recreational and/or medicinal manner. However, although the literature has already described the presence of endocannabinoid system components since the early stages of CNS development, many of their physiological effects during this stage have not yet been established. Moreover, it is still uncertain how the endocannabinoid system can be altered in terms of cell proliferation and cell fate, neural migration, neural differentiation, synaptogenesis and particularly cell death. In relation to cell death in the CNS, knowledge about the effects of cannabinoids is scarce. Thus, the present work aims to review the role of the endocannabinoid system in different aspects of CNS development and discuss possible side effects or even opportunities for treating some conditions in the development of this tissue.
 
Multiple sclerosis (MS) is an inflammatory-demyelinating disease of the central nervous system (CNS) mediated by aberrant auto-reactive immune responses. The current immune-modulatory therapies are unable to protect and repair immune-mediated neural tissue damage. One of the therapeutic targets in MS is the sphingosine-1-phosphate (S1P) pathway which signals via sphingosine-1-phosphate receptors 1–5 (S1P1-5). S1P receptors are expressed predominantly on immune and CNS cells. Considering the potential neuroprotective properties of S1P signaling, we utilized S1P1-GFP (Green fluorescent protein) reporter mice in the cuprizone-induced demyelination model to investigate in vivo S1P – S1P1 signaling in the CNS. We observed S1P1 signaling in a subset of neural stem cells in the subventricular zone (SVZ) during demyelination. During remyelination, S1P1 signaling is expressed in oligodendrocyte progenitor cells in the SVZ and mature oligodendrocytes in the medial corpus callosum (MCC). In the cuprizone model, we did not observe S1P1 signaling in neurons and astrocytes. We also observed β-arrestin-dependent S1P1 signaling in lymphocytes during demyelination and CNS inflammation. Our findings reveal β-arrestin-dependent S1P1 signaling in oligodendrocyte lineage cells implying a role of S1P1 signaling in remyelination. Graphical Abstract
 
Mechanisms of rTMS in synaptic plasticity after ischemic stroke. Several molecules play important roles in the modulation of synaptic plasticity. After ischemic stroke, rTMS can enhance the levels of synaptic plasticity-associated genes, such as Dlx6, Calb2, Zic1, Crhr2, and Gng4, and Grin3a; rTMS can also inhibit the overexpression of pro-inflammatory cytokines TNF, and facilitate the expression of anti-inflammatory cytokines IL-10 to improve the environment of synaptic growth; In addition, rTMS-induced MeCP2 phosphorylation facilitated the expression of BDNF and the interaction between BDNF exon IV and RACK1. Ultimately, those proteins and genes can improve the structure and function of synapses by up-regulating the levels of PSD 95, mGlu 2/3R, and synapsin-1. rTMS repetitive transcranial magnetic stimulation, NMDARN-methyl-d-aspartic acid receptors, BDNF brain-derived neurotrophic factor, PSD 95 post-synaptic density protein 95, mGlu2/3R glutamate receptor 2/3, MeCP2 methyl CpG binding protein 2, RACK receptor for activated C kinase 1, GLU glutamate
Mechanisms of rTMS in BBB after ischemic stroke. rTMS can facilitate the ischemia-induced increase of HIF-1α and A1 shift to A2 in vessel-associated astrocytes. The increased HIF-1α can promote the release of angiogenesis-related factors TGFβ and VEGF in A2 astrocytes. In addition, TMS can also facilitate the secretion of CGRP, increase the activity of Bcl-xL, and decrease the activation of Bax, caspase-1, caspase-3. Ultimately, those molecules can improve BBB function by mitigating BBB permeabilization by enhancing the expression of claudin-5, ZO-1, occludin, and caveolin-1. BBB blood–brain barrier disruption, HIF-1α hypoxia-inducible factor-1α, CGRP calcitonin gene-related peptide, VEGF vascular endothelial growth factor
Stroke is a common cerebrovascular disease with high morbidity, mortality, and disability worldwide. Post-stroke dysfunction is related to the death of neurons and impairment of synaptic structure, which results from cerebral ischemic damage. Currently, transcranial magnetic stimulation (TMS) techniques are available to provide clinically effective interventions and quantitative diagnostic and prognostic biomarkers. The development of TMS has been 40 years and a range of repetitive TMS (rTMS) protocols are now available to regulate neuronal plasticity in many neurological disorders, such as stroke, Parkinson disease, psychiatric disorders, Alzheimer disease, and so on. Basic studies in an animal model with ischemic stroke are significant for demonstrating potential mechanisms of neural restoration induced by rTMS. In this review, the mechanisms were summarized, involving synaptic plasticity, neural cell death, neurogenesis, immune response, and blood–brain barrier (BBB) disruption in vitro and vivo experiments with ischemic stroke models. Those findings can contribute to the understanding of how rTMS modulated function recovery and the exploration of novel therapeutic targets. Graphical Abstract The mechanisms of rTMS in treating ischemic stroke from animal models. rTMS can prompt synaptic plasticity by increasing NMDAR, AMPAR and BDNF expression; rTMS can inhibit pro-inflammatory cytokines TNF and facilitate the expression of anti-inflammatory cytokines IL-10 by shifting astrocytic phenotypes from A1 to A2, and shifting microglial phenotypes from M1 to M2; rTMS facilitated the release of angiogenesis-related factors TGFβ and VEGF in A2 astrocytes, which can contribute to vasculogenesis and angiogenesis; rTMS can suppress apoptosis by increasing Bcl-2 expression and inhibiting Bax, caspase-3 expression; rTMS can also suppress pyroptosis by decreasing caspase-1, IL-1β, ASC, GSDMD and NLRP1 expression. rTMS, repetitive transcranial magnetic stimulation; NMDAR, N-methyl-D-aspartic acid receptors; AMPAR: α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; BDNF, brain-derived neurotrophic factor; VEGF, vascular endothelial growth factor; GSDMD: cleaved Caspase-1 cleaves Gasdermin D; CBF: cerebral blood flow.
 
Neuroanatomy of the olfactory system. Volatile chemicals (odorants) enter the nasal cavity and are detected by olfactory sensory neurons (microvillar cells, blue, and olfactory sensory neurons, purple) in the main olfactory epithelium. Olfactory sensory axons enter the cribriform plate into the glomerular layer of the olfactory bulb where they synapse with periglomerular cells. The signal is propagated to the cell bodies of tufted cells in the external plexiform layer and mitral cells in the mitral cell layer. Finally, the neurons in the external plexiform and mitral layer send excitatory projections to the different areas of the primary olfactory cortex (Amygdala, Lateral entorhinal cortex, Olfactory tubercle, Piriform cortex, Anterior olfactory nucleus), via the lateral olfactory tract where the integration of olfactory information takes place. The system is mainly connected with the hypothalamus via poly-synaptic pathways from the olfactory bulb to the preoptic area of the hypothalamus. Other poly-synaptic pathways connecting the olfactory system with the paraventricular nucleus, dorsomedial nucleus, ventromedial nucleus and lateral hypothalamic area have also been described (see the text). Created with BioRender.com
Olfactory-hypothalamic axis role in metabolic balance. a Food odors are perceived through the sensory neurons in the olfactory epithelium (OE) which project to the olfactory bulb (OB). Here the mitral (black) and tufted (pink) cells send their projections to the anterior olfactory nucleus (AON), the olfactory tubercle (OT) and piriform cortex (PC), and to other parts of the olfactory cortex, in this region the olfactory information is processed for odor detection, discrimination, learning and recalling, these processes also include other cortexes and the hippocampus (not shown here). In a separated pathway, many regions in the olfactory cortex (see text) send axonal projections to the preoptic (POA) and the lateral (LH) areas of the hypothalamus, that in turn establish connections with nuclei involved in the regulation of food intake and energy balance, like the dorsomedial hypothalamus, the paraventricular nucleus, the ventromedial hypothalamus and the arcuate nucleus (ARC). Hypothetically, olfactory information could reach the ARC, where food perception activates the POMC anorexigenic neurons to reduce food intake and increase energy expenditure, and at the same time the olfactory pathway would also inhibit the orexigenic AgRP/NPY neurons. Peripheral hormones like insulin and leptin, are known signals that activate POMC neurons in hypothalamus thus inhibiting food intake and increasing energy expenditure, and in the olfactory system it is known that these hormones are able to suppress olfactory sensitivity. The pathway activated when the individuals have a normal weight is represented in blue and its alteration in obesity is represented in faded red, in which hormones like insulin and leptin do not exert its actions nor in the olfactory system neither in the ARC thus the olfactory perception remains hyperactivated and the ARC circuits are unable to suppress food nor to elicit energy expenditure. b Hypothetical model of how the “olfactory-hypothalamic axis” could be intervened intranasally through the direct activation or desensitization of the olfactory system to induce the activation of neurons in the ARC that inhibit food intake and promote the activation of the autonomic mechanisms involved in the increase of energy expenditure. Created with BioRender.com
The olfactory system is responsible for the reception, integration and interpretation of odors. However, in the last years, it has been discovered that the olfactory perception of food can rapidly modulate the activity of hypothalamic neurons involved in the regulation of energy balance. Conversely, the hormonal signals derived from changes in the metabolic status of the body can also change the sensitivity of the olfactory system, suggesting that the bidirectional relationship established between the olfactory and the hypothalamic systems is key for the maintenance of metabolic homeostasis. In the first part of this review, we describe the possible mechanisms and anatomical pathways involved in the modulation of energy balance regulated by the olfactory system. Hence, we propose a model to explain its implication in the maintenance of the metabolic homeostasis of the organism. In the second part, we discuss how the olfactory system could be involved in the development of metabolic diseases such as obesity and type two diabetes and, finally, we propose the use of intranasal therapies aimed to regulate and improve the activity of the olfactory system that in turn will be able to control the neuronal activity of hypothalamic centers to prevent or ameliorate metabolic diseases.
 
Excessive mitophagy plays a role in neuronal death in spinal cord injury (SCI), its molecular regulation remains largely unknown. The present study aims to determine the role of NIX, a member of a unique subfamily of death-inducing mitochondrial proteins, in the regulation of mitophagy in SCI. Here we show that NIX is highly upregulated in SCI and hypoxia, and localized to mitochondria. The mitochondria-bound NIX interacts with autophagosome-localized LC3 (Microtubule-associated protein 1 light chain 3) to form a mitochondria-NIX-LC3-autophagosome complex, resulting in excessive mitophagy in SCI. Downregulation of NIX by RNA interference restores the function of mitochondria in spinal cord neurons under hypoxia. Importantly, inhibition of NIX improves recovery of locomotor function in rats after SCI. The present study demonstrates that NIX interacts with LC3 to activate excessive mitophagy in SCI. Inhibition of NIX is therefore likely a neuroprotective strategy.
 
Dexamethasone (DEX) is frequently used to treat women at risk of preterm delivery, but although indispensable for the completion of organ maturation in the fetus, antenatal DEX treatment may exert adverse sex-dimorphic neurodevelopmental effects. Literature findings implicated oxidative stress in adverse effects of DEX treatment. Purinergic signaling is involved in neurodevelopment and controlled by ectonucleotidases, among which in the brain the most abundant are ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5ʹ-nucleotidase (e5ʹNT/CD73), which jointly dephosphorylate ATP to adenosine. They are also involved in cell adhesion and migration, processes integral to brain development. Upregulation of CD39 and CD73 after DEX treatment was reported in adult rat hippocampus. We investigated the effects of maternal DEX treatment on CD39 and CD73 expression and enzymatic activity in the rat fetal brain of both sexes, in the context of oxidative status of the brain tissue. Fetuses were obtained at embryonic day (ED) 21, from Wistar rat dams treated with 0.5 mg DEX/kg/day, at ED 16, 17, and 18, and brains were processed and used for further analysis. Sex-specific increase in CD39 and CD73 expression and in the corresponding enzyme activities was induced in the brain of antenatally DEX-treated fetuses, more prominently in males. The oxidative stress induction after antenatal DEX treatment was confirmed in both sexes, although showing a slight bias in males. Due to the involvement of purinergic system in crucial neurodevelopmental processes, future investigations are needed to determine the role of these observed changes in the adverse effects of antenatal DEX treatment.
 
Glioblastoma multiforme (GBM) is account for 70% of all primary malignancies of the central nervous system. The median survival of human patients after treatment is around 15 months. There are several biological targets which have been reported that can be pursued using ligands with varied structures to treat this disease. In our group, we have developed several ligands that target a wide range of proteins involved in anticancer effects, such as histone deacetylase (HDACs), G protein-coupled estrogen receptor 1 (GPER), estrogen receptor-beta (ERβ) and NADPH oxidase (NOX), that were screened on bidimensional (2D) and tridimensional (3D) GBM stem cells like (GSC). Our results show that some HDAC inhibitors show antiproliferative properties at 21–32 µM. These results suggest that in this 3D culture, HDACs could be the most relevant targets that are modulated to induce the antiproliferative effects that require in the future further experimental studies.
 
Deleterious effects of ischemia on presynaptic and postsynaptic neurons. The interruption or reduction of blood flow is associated with the decrease of O2 levels and nutrient supply. Neurons respond to these effects by decreasing aerobic glycolysis, while increasing anaerobic glycolytic process, leading to the accumulation of lactate and to a pH reduction. As ATP levels decrease (1 and I), the failure of Na⁺/K⁺/ATP pumps may occur (2 and II), which cause electrolyte imbalance (3 and III), depolarization and opening of Ca²⁺ voltage-dependent membrane channels (4 and IV). In the presynaptic neuron, these alterations increase neurotransmitter release, especially glutamate (5). The reversal of EAAT transporters contributes to the increase in glutamate availability in the synaptic cleft as well (6). ATP deficiency also impacts neurons by generating reactive oxygen/nitrogen species (e.g., superoxide and peroxynitrite). The depolarization mediated by intracellular sodium increase (III) stimulates voltage-gated Ca²⁺ channel (IV). Intracellular Ca²⁺ level is also elevated through the reversal of Na⁺/Ca²⁺ exchanger (V). Ca²⁺ overload also affects the postsynaptic neuron as a result of NMDA receptor hyperactivation (VI), triggering glutamate excitotoxicity. Thus, intracellular Ca²⁺ accumulation leads to the activation of different death pathways such as the one mediated by NOS, Calpain, Caspase, and phospholipase A2 (PLA2). It is important to note that other pathways contributing to cell death are not described in the scheme for summarization purposes. For the clarity of the scheme Bax/Bad are show in mitochondria matrix
Temporal profile of glutamate release/excitotoxicity (green), oxidative stress (blue), inflammation (orange), and cell death (purple) after ischemia induction. Glutamate release begins a few minutes after the onset of ischemia, reaching a peak within an hour if the noxious event lasts for that long. Extracellular glutamate content gradually decreases as soon as reperfusion takes place, and the time to restore basal levels is related to the severity of ischemia. Excitotoxic and necrotic cell death occurs rapidly at the ischemic core but programmed cell death and infarct volume are still ongoing for some days until it is not detectable anymore in weeks. At the onset of reperfusion, with the reestablishment of oxygen supply, reactive oxygen species production dramatically increases, reaching a peak close to 24 h after ischemia, when it starts to decline. Inflammation is the latest event, with microglial and macrophage activation, adhesion molecules expression, neutrophil infiltration, astroglial response, and cytokine release taking place within some hours after ischemia. Since inflammation is a multifactorial process, different phenomena occur in maximal intensity in a larger time window, with some events still rising up to 7 days, decreasing thereafter. The curves for non-treated conditions were based on data that explore the temporal pattern of mentioned parameters in the same study. The effect of adenosinergic intervention was based on data from studies mentioned throughout the text that explored at least one of the illustrated events in ischemia and demonstrated protective effects at one or more of the time periods shown
Intracellular pathways coupled to adenosine receptors and CNS distribution. a There are four types of adenosine receptors named A1, A2A, A2B, and A3. The A1 and A3 receptors activate Gi/o protein, while A2A and A2B receptors are coupled to Gs/olf protein inhibiting and stimulating, respectively, adenylyl cyclase. Thus, adenosine receptors regulate cAMP levels, which impacts on protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) activity. A series of other effector proteins may also be modulated. Moreover, adenosine receptors can stimulate the phospholipase C (PLC) pathway. A1 receptors regulate PLC via beta/gamma complex (Biber et al. 1997; Dickenson and Hill 1998), whereas A2A receptors act through Gq protein (Ribeiro et al. 2016; Socodato et al. 2011). Both A2B and A3 receptors can also stimulate PLC (Abbracchio et al. 1995; Kohno et al. 1996; Pilitsis and Kimelberg 1998). b The distribution of adenosine receptors varies dramatically within the CNS. High densities of A1 receptors are expressed in the cortex, hippocampus, and cerebellum, while A2A receptors are more abundant in the striatum and olfactory bulb. In contrast, A3 and A2B receptors are diffusely distributed in all brain regions in smaller amounts when compared to A1 and A2A receptors (Sheth et al. 2014). A1, A2A, A2B, and A3 receptors are also found in retinal cells, a structure that is part of the CNS, of different animals (Dos Santos-Rodrigues et al. 2015; Brito et al. 2016; Grillo et al. 2019; Portugal et al. 2021)
Effects of adenosine receptor modulation in ischemic events. a The increase of extracellular adenosine availability during ischemia allows the activation of all adenosine receptors in different cell types. In astrocytes, stimulation (positive symbol, +) of A1 receptors (green), or inhibition (─┤) of A2A receptors (red), reduces EAAT1/2 exacerbating the augment in extracellular glutamate and contributing to excitotoxicity. Astrocytes and microglia experience an increase in CD39 and CD73 content in ischemic events. In the case of A1 receptors, the regulation of EAATs occurs through MAPK/PKC pathway. The activation of microglial A2A receptors induces intracellular pathways related to an inflammatory response. Furthermore, in endothelial cells, A2B (purple) and A3 (yellow) receptors stimulation, or A2A receptor inhibition, reduces VCAM/ICAM content, immune cells infiltration, BBB breakdown, and edema. The inhibition of A2A receptors in oligodendrocyte and microglia cells reduces, respectively, p-JNK and p-p38 as well as TNF-α. Canonical pathway is represented in postsynaptic neuron. The right panel depicts the pre- and postsynaptic terminals and the effect of adenosine receptors agonists or antagonists during ischemia. b The activation of presynaptic A1 receptors decreases glutamate release through a direct mechanism or through the inhibition of voltage-gated Ca²⁺ channels (VGCC). Antagonists of A2A presynaptic receptors also inhibit glutamate release. In the postsynaptic neurons, A1 agonism or A2A antagonism triggers multiple intracellular pathways promoting antioxidant and anti-inflammatory responses, decreasing oxidative stress and cell death
Ischemia is characterized by a transient, insufficient, or permanent interruption of blood flow to a tissue, which leads to an inadequate glucose and oxygen supply. The nervous tissue is highly active, and it closely depends on glucose and oxygen to satisfy its metabolic demand. Therefore, ischemic conditions promote cell death and lead to a secondary wave of cell damage that progressively spreads to the neighborhood areas, called penumbra. Brain ischemia is one of the main causes of deaths and summed with retinal ischemia comprises one of the principal reasons of disability. Although several studies have been performed to investigate the mechanisms of damage to find protective/preventive interventions, an effective treatment does not exist yet. Adenosine is a well-described neuromodulator in the central nervous system (CNS), and acts through four subtypes of G-protein-coupled receptors. Adenosine receptors, especially A1 and A2A receptors, are the main targets of caffeine in daily consumption doses. Accordingly, caffeine has been greatly studied in the context of CNS pathologies. In fact, adenosine system, as well as caffeine, is involved in neuroprotection effects in different pathological situations. Therefore, the present review focuses on the role of adenosine/caffeine in CNS, brain and retina, ischemic events.
 
Construction of a transwell astrocytes–neuron co-culture system. a Graphic presentation of the transwell astrocytes–neuron co-culture system. b Neuron monolayer immunofluorescence staining of GFAP (astrocyte), MAP2 (neurite), and DAPI (nucleus) after transwell co-culture. Scale bar = 20 μm. c Statistic graphs showing amounts of penetrated astrocytes (left) and ratio of astrocytes/neuron (right) in 3, 6, 12, 24, 36 h after transwell co-culture. Bar graphs indicate mean ± SD
GJA1-20K enhanced protection and recovery of damaged neurons. a Western blotting analysis of total and phosphorylated Cx43 after transfection of GJA1-20K in astrocytes. GJA1-20K were transfected with a C-terminal HA tag. As GJA1-20K shares the same amino acids with Cx43 C-terminus, Cx43 N-terminus antibodies and HA antibodies were used to target Cx43 and GJA1-20K, respectively. b Graph-scale quantification analysis of WB results, compared with vehicle control group. **P < 0.01, ***P < 0.001 (c) MAP2 immunofluorescence staining of separated cultured and co-cultured astrocytes and neurons in transwell system. Single-cultured normal and damaged neurons, and damaged neurons co-cultured with vehicle/GJA1-20K astrocytes were assessed. Scale bar = 20 μm. d Statistics of dendrite length of MARP2 staining results. **P < 0.01, versus normal group; #P < 0.05, ##P < 0.01, n.s., not significant, versus single-cultured damaged group. Bar graphs indicate mean ± SD
GJA1-20K promoted mitochondria transmission from astrocytes to neurons. a Immunofluorescence staining of neuron–astrocyte co-cultures with or without Gap26 treatment. Astrocytes were pre-labeled with MitoTracker before transwell co-culture. Gap26 were introduced to block Cx43. b Quantification analysis of immunofluorescence intensity of transferred astrocyte mitochondria. **P < 0.01, ***P < 0.01, versus vehicle group. (C) Statistics of neurons with transferred astrocyte mitochondria. **P < 0.01, ***P < 0.01, versus vehicle group. d Statistics of relative dendrite length of neurons with or without delivered astrocyte mitochondria. *P < 0.05, **P < 0.01, versus neurons without astrocytic mitochondria; #P < 0.05, versus vehicle group. Bar graphs indicate mean ± SD
Astrocyte GJA1-20K induced stability and activity of neuronal mitochondria via transwell co-culture. a Western blotting analysis of two crucial activators of mitochondria biosynthesis, mtTFA and PGC-1a. Quantification was shown in the lower panel. b Western blotting analysis of Tom20 and mtCO2. Quantification was shown in the lower panel. c qPCR analysis of mRNA levels of above four protein factors. Bar graphs indicate mean ± SD. *P < 0.05, **P < 0.01, versus single-cultured damaged neurons; #P < 0.05, versus vehicle transfected group
Astrocytes are crucial in neural protection after traumatic brain injury (TBI), a global health problem causing severe brain tissue damage. Astrocytic connexin 43 (Cx43), encoded by GJA1 gene, has been demonstrated to facilitate the protection of astrocytes to neural damage with unclear mechanisms. This study aims to explore the role of GJA1-20K/Cx43 axis in the astrocyte–neuron interaction after TBI and the underlying mechanisms. Primarily cultured cortical neurons isolated from embryonic C57BL/6 mice were treated by compressed nitrogen–oxygen mixed gas to simulate TBI-like damage in vitro. The transwell astrocyte–neuron co-culture system were constructed to recapitulate the interaction between the two cell types. Quantitative PCR was applied to analyze mRNA level of target genes. Western blot and immunofluorescence were conducted to detect target proteins expression. GJA1-20K overexpression significantly down-regulated the expression of phosphorylated Cx43 (p-Cx43) without affecting the total Cx43 protein level. Besides, GJA1-20K overexpression obviously enhanced the dendrite length, as well as the expression levels of function and synthesis-related factors of mitochondria in damaged neurons. GJA1-20K up-regulated functional Cx43 expression in astrocytes, which promoted mitochondria transmission from astrocytes to neurons which might be responsible to the protection of astrocyte to neurons after TBI-like damage in vitro.
 
The present research has reported that cannabinoid receptor 1 (CB1) agonist, delta-(9)-tetrahydrocannabinol (THC) modulates synaptogenesis during overexcitation. Microtubule and synaptic distribution, poly(ADP)-ribose (PAR) accumulation were estimated during overexcitation and in the presence of THC. Low concentration of THC (10 nM) increased synaptophysin expression and neurite length, while high concentration of THC (1 µM) induced neurotoxicity. Glutamate caused the loss of neurons, reducing the number and the length of neurites. The high concentration of THC in the presence of glutamate caused the PAR accumulation in the condensed nuclei. Glutamate upregulated genes that are involved in synaptogenesis and excitatory signal cascade. Glutamate downregulated transcription of beta3 tubulin and microtubule-associated protein 2. THC partially regulated gene expression that is implicated in the neurogenesis and excitatory pathways. This suggests that CB1 receptors play a role in neurite growth and the low concentration of THC protects neurons during overexcitation, whereas the high concentration of THC enhances the neurotoxicity.
 
Neurons in the penumbra (the area surrounding ischemic tissue that consists of still viable tissue but with reduced blood flow and oxygen transport) may be rescued following stroke if adequate perfusion is restored in time. It has been speculated that post-stroke angiogenesis in the penumbra can reduce damage caused by ischemia. However, the mechanism for neovasculature formation in the brain remains unclear and vascular-targeted therapies for brain ischemia remain suboptimal. Here, we show that VEGFR1 was highly upregulated in pericytes after stroke. Knockdown of VEGFR1 in pericytes led to increased infarct area and compromised post-ischemia vessel formation. Furthermore, in vitro studies confirmed a critical role for pericyte-derived VEGFR1 in both endothelial tube formation and pericyte migration. Interestingly, our results show that pericyte-derived VEGFR1 has opposite effects on Akt activity in endothelial cells and pericytes. Collectively, these results indicate that pericyte-specific expression of VEGFR1 modulates ischemia-induced vessel formation and vascular integrity in the brain.
 
Proteomic analysis. a Statistical analysis of proteins based on LFQ of both SZ and control group. b Two-sample test results (statistically significant results considering a P-value ≤ 0.05). c STRING network analysis with protein–protein interaction mapping. d Hierarchal clustering heatmap of proteins identified and selected by Perseus software. Up-regulated proteins are represented in blue and down-regulated proteins in brown
Comparative analysis and representations of proteins and ECM model. a Comparative analysis of quantitative results obtained by a western blot. Statistical significance was found for fibronectin (P = 0.0166), SPARC (P = 0.0003), lumican (P = 0.0012), and nidogen-1 (P < 0.0001). b Protein expression of both SZ and control groups, and structures of proteins using the SWISS-MODEL online platform (https://swissmodel.expasy.org). c Structural model of ECM with fibronectin, SPARC, nidogen-1, and lumican. d Protein expression in CSF samples of controls to prove their presence in the CNS. BBB—blood–brain barrier. e Fluorescence microscopy images of PFC sections of a patients with SZ and a healthy control labelled by fibronectin, lumican, nidogen-1, and SPARC, and a positive control (GFAP). Confocal images of PFC sections labelled by anti-fibronectin, anti-lumican, anti-nidogen-1, and anti-SPARC antibodies (green) and anti-GFAP positive control (red) showing the distribution of ECM proteins in the PFC of a patient with SZ and a healthy control
β-III tubulin and caspase 3 expressions in PFC sections. Immunofluorescence staining using β-III tubulin as a marker of neuronal identity and caspase 3 as a marker of apoptosis. In the PFC sections of the patients with SZ, there was an activation of caspase 3 suggesting apoptosis of neurons
Correlation between plasma levels of proteins and clinical variables
The brain extracellular matrix (ECM) is involved in crucial processes of neural support, neuronal and synaptic plasticity, extrasynaptic transmission, and neurotransmission. ECM is a tridimensional fibrillary meshwork composed of macromolecules that determine its bioactivity and give it unique characteristics. The characterization of the brain ECM is critical to understand its dynamic in SZ. Thus, a comparative study was developed with 71 patients with schizophrenia (SZ) and 70 healthy controls. Plasma of participants was analysed by label-free liquid chromatography–tandem mass spectrometry, and the results were validated using the classical western blot method. Lastly, immunostaining of post-mortem human brain tissue was performed to analyse the distribution of the brain ECM proteins by confocal microscopy. The analysis identified four proteins: fibronectin, lumican, nidogen-1, and secreted protein acidic and rich in cysteine (SPARC) as components of the brain ECM. Statistical significance was found for fibronectin (P = 0.0166), SPARC (P = 0.0003), lumican (P = 0.0012), and nidogen-1 (P < 0.0001) that were decreased in the SZ group. Fluorescence imaging of prefrontal cortex (PFC) sections revealed a lower expression of ECM proteins in SZ. Our study proposes a pathophysiological dysregulation of proteins of the brain ECM, whose abnormal composition leads to a progressive neuronal impairment and consequently to neurodegenerative processes due to lack of neurophysiological support and dysregulation of neuronal homeostasis. Moreover, the brain ECM and its components are potential pharmacological targets to develop new therapeutic approaches to treat SZ.
 
Dendrogram analysis of SNP–SNP interaction (NDRG1). The colors in the tree diagram represent synergy (yellow) or redundancy (blue)
Haplotype block map for 2 SNPs in NDRG1 gene. The numbers inside the diamonds indicate the D′ for pairwise analyses
Kaplan–Meier curves for overall survival and progression-free survival according to the glioma patients with different clinical factors (a OS according to surgical operation; b OS according to chemotherapy status; c PFS according to surgical operation; d PFS according to radiotherapy status)
Kaplan–Meier curves for overall survival and progression-free survival according to the astrocytoma patients with different clinical factors (a OS according to surgical operation; b PFS according to surgical operation; c PFS according to radiotherapy status)
Glioma patient survival based on NDRG1 rs3779941 polymorphism. Kaplan–Meier survival curves are plotted for overall and progression-free survival (a OS based on NDRG1 rs3779941 polymorphism; b PFS based on NDRG1 rs3779941 polymorphism)
Glioma is a highly fatal malignant tumor with a high recurrence rate, poor clinical treatment effect, and prognosis. We aimed to determine the association between single nucleotide polymorphisms (SNPs) of NDRG1 and glioma risk and prognosis in the Chinese Han population. 5 candidate SNPs were genotyped by Agena MassARRAY in 558 cases and 503 controls; logistic regression was used to analyze the relationship between SNPs and glioma risk. We used multi-factor dimensionality reduction to analyze the interaction of ‘SNP–SNP’; the prognosis analysis was performed by log-rank test, Kaplan–Meier analysis, and Cox regression model. Our results showed that the polymorphisms of rs3808599 was associated with the reduction of glioma risk in all participants (OR 0.41, p = 0.024) and the participants ≤ 40 years old (OR 0.30, p = 0.020). rs3802251 may reduce glioma risk in all participants (OR 0.79, p = 0.008), the male participants (OR 0.68, p = 0.033), and astrocytoma patients (OR 0.81, p = 0.023). rs3779941 was associated with poor glioma prognosis in all participants (HR = 2.59, p = 0.039) or astrocytoma patients (HR = 2.63, p = 0.038). We also found that the key factors for glioma prognosis may include surgical operation, radiotherapy, and chemotherapy. This study is the first to find that NDRG1 gene polymorphisms may have a certain association with glioma risk or prognosis in the Chinese Han population.
 
Recent evidences have shown the therapeutic potential of transcranial photobiomodulation on traumatic brain injury and Alzheimer’s disease. Despite the promising benefits in the brain, little is known about the laser’s effects in the absence of pathological conditions. We submitted young (4 months old) and aged (20 months old) rats to transcranial low-level laser and evaluated their exploratory activity and habituation in open field, anxiety in elevated plus maze, spatial memory in Barnes maze, and aversive memory in a step-down inhibitory avoidance task. Additionally, the levels of a panel of inflammatory cytokines and chemokines were quantified in two different brain regions: the cerebral cortex and the hippocampus. Young and aged rats submitted to transcranial laser exhibited better cognitive performance in Barnes maze than did control rats. Transcranial laser therapy decreased cortical levels of GM-CSF, IL-10, MCP-1, LIX, and TNFα in young rats and IL-5 in aged rats. High levels of IL-6, IL-10, and TNF-alpha were found in the cerebral cortex of aged rats submitted to transcranial laser. In the hippocampus, a decrease in IP-10 and fractalkine levels was observed in the aged rats from the laser group when compared to the aged rats from the control group. Our data indicate that transcranial photobiomodulation improves spatial learning and memory and alters the neuroinflammatory profile of young and aged rats’ brains.
 
Angiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.
 
Ischemic preconditioning (IPC) is an approach of protection against cerebral ischemia by inducing endogenous cytoprotective machinery. However, few studies in neurogenesis and oligodendrogenesis after IPC have been reported, especially the latter. The purpose of this study is to test our hypothesis that IPC may also induce cell proliferation and oligodendrogenesis in the subventricular zone and striatum, as well as to investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) on oligodendrogenesis. IPC was induced in mice by 12-min ischemia through the occlusion of the middle cerebral artery. Newly generated cells were labeled with 5-bromo-2′-deoxyuridine. Our findings demonstrated that IPC stimulated the proliferation of neural stem cells in the subventricular zone, promoted the generation of oligodendrocyte precursor cells in the striatum and corpus callosum/external capsule (CC/EC), and stimulated oligodendrocyte precursor cells differentiation into oligodendrocytes in the striatum and the CC/EC. Furthermore, we describe a crucial role for Nrf2 in IPC-induced oligodendrogenesis in the subventricular zone, striatum, and CC/EC and show for the first time that Nrf2 promoted the migration and differentiation of oligodendrocyte precursor cells into oligodendrocytes in the striatum and CC/EC. Our data imply that IPC stimulates the oligodendrogenesis in the brain and that Nrf2 signaling may contribute to the oligodendrogenesis.
 
Single-nucleotide variant (SNV) is a single base mutation at a specific location in the genome and may play an import role in epilepsy pathophysiology. The aim of this study was to review case–control studies that have investigated the relationship between SNVs within microRNAs (miRs) sequences or in their target genes and epilepsy susceptibility from January 1, 2010 to October 31, 2020. Nine case–control studies were included in the present review. The mainly observed SNVs associated with drug-resistant epilepsy (DRE) risk were SNVs n.60G > C (rs2910164) and n.-411A > G (rs57095329), both located at miR-146a mature sequence and promoter region, respectively. In addition, the CC haplotype (rs987195-rs969885) and the AA genotype at rs4817027 in the MIR155HG/miR-155 tagSNV were also genetic susceptibility markers for early-onset epilepsy. MiR-146a has been observed as upregulated in human astrocytes in epileptogenesis and it regulates inflammatory process through NF-κB signaling by targeting tumor necrosis factor-associated factor 6 (TRAF6) gene. The SNVs rs2910164 and rs57095329 may modify the expression level of mature miR-146a and the risk for epilepsy and SNVs located at rs987195-rs969885 haplotype and at rs4817027 in the MIR155HG/miR-155 tagSNV could interfere in the miR-155 expression modulating inflammatory pathway genes involved in the development of early-onset epilepsy. In addition, SNVs rs662702, rs3208684, and rs35163679 at 3′untranslated region impairs the ability of miR-328, let-7b, and miR-200c binding affinity with paired box protein PAX-6 (PAX6), BCL2 like 1 (BCL2L1), and DNA methyltransferase 3 alpha (DNMT3A) target genes. The SNV rs57095329 might be correlated with DRE when a larger number of patients are evaluated. Thus, we concluded that the main drawback of most of studies is the small number of individuals enrolled, which lacks sample power.
 
Despite the widespread use of the SH-SY5Y human neuroblastoma cell line in modeling human neurons in vitro, protocols for growth, differentiation and experimentation differ considerably across the literature. Many studies fully differentiate SH-SY5Y cells before experimentation, to investigate plasticity measures in a mature, human neuronal-like cell model. Prior to experimentation, serum is often removed from cell culture media, to arrest the cell growth cycle and synchronize cells. However, the exact effect of this serum removal before experimentation on mature, differentiated SH-SY5Y cells has not yet been described. In studies using differentiated SH-SY5Y cells, any effect of serum removal on plasticity markers may influence results. The aim of the current study was to systematically characterize, in differentiated, neuronal-like SH-SY5Y cells, the potentially confounding effects of complete serum removal in terms of morphological and gene expression markers of plasticity. We measured changes in commonly used morphological markers and in genes related to neuroplasticity and synaptogenesis, particularly in the BDNF-TrkB signaling pathway. We found that complete serum removal from already differentiated SH-SY5Y cells increases neurite length, neurite branching, and the proportion of cells with a primary neurite, as well as proportion of βIII-Tubulin and MAP2 expressing cells. Gene expression results also indicate increased expression of PSD95 and NTRK2 expression 24 h after serum removal. We conclude that serum deprivation in differentiated SH-SY5Y cells affects morphology and gene expression and can potentially confound plasticity-related outcome measures, having significant implications for experimental design in studies using differentiated SH-SY5Y cells as a model of human neurons.
 
5-HT Synthesis and Metabolism. The essential amino acid, tryptophan, is the sole precursor molecule involved with the synthesis of 5-HT. Tryptophan must first cross the BBB to enter the CNS prior to 5-HT synthesis. Once in the CNS, l-tryptophan is hydroxylated to 5-hydroxytryptophan (5-HTP) by the enzyme tryptophan hydroxylase type 2 (TPH2). This is followed by subsequent decarboxylation involving the enzyme l-aromatic acid decarboxylase (AADC) transforming 5-HTP into 5-HT. This fully synthesized 5-HT is then taken up into vesicles in the axon terminal via vesicular monoamine transporter isoform 2 (VMAT2). Following an action potential, 5-HT is released into the synapse. It can interact with both presynaptic and postsynaptic receptors. In addition, 5-HT can be metabolized by monoamine oxidase-A (MAO-A) and aldehyde dehydrogenase to the metabolite 5-hydroxyindoleacetic acid (5-HIAA). 5-HT synthesis is regulated by the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase, and the cofactor of tryptophan hydroxylase, tetrahydrobiopterin. All 5-HTRs are heteroreceptors and postsynaptically expressed on non-serotonergic neurons and autoreceptors located presynaptically on the serotonergic neurons. SERTs are localized on the axon terminal and soma of the serotonergic neurons. SERT undergoes conformational modifications and transfers one or more molecules in each cycle (El-Merahbi et al. 2015; Sahu et al. 2018)
of proposed signaling mediated by 5-HT receptor subtypes. 5-HT1A, B, D, E, F, 5-HT2A,B,C, 5-HT4, 5-HT5A, B, 5-HT6, and 5-HT7 receptors are classified as G protein-coupled receptors (GPCRs), while 5-HT3A, B, C, D, E receptors are ligand-gated ion channels (LGIC). The serotonergic GPCRs are made of a common structure and contain seven transmembrane alpha-helices (7-TM), which are connected by three extracellular and three intracellular loop. Upon ligand binding, the intracellular loop and C-terminal tail interact with specific G protein families, including Gαs, Gαi/o, Gαq/11, and followed by the generation of second messengers. 5-HT1Rs couple to Gαi/o proteins, 5-HT2Rs couple to Gαq/11 proteins, and 5-HT4, 5-HT6 and 5-HT7 receptors couple to Gαs proteins. Although no primary mechanistic pathway has been recognized for 5-HT5Rs, there are reports suggesting that it couples to Gαi/o. 5-HT3Rs are made of five monomers (A to E) and form a tube-like channel. These GPCRs work through a variety of intracellular functional proteins, including adenylyl cyclase (AC), Phospholipase-C (PLC), voltage-gated N-type Ca²⁺ channels, and hyperpolarizing K⁺ channels. 5-HT1Rs inhibit neuronal activity by suppressing the activity of AC, and decreasing the cAMP level, and activate MAPK pathway. Coupling through 5-HT2Rs induces PLC activity which hydrolyze membrane phospholipids to release second messengers, such as inositol 1,4,5-triphosphate (IP3) and diacylglycerols (DAGs). It also stimulates protein kinase C (PKC), MAPK pathway, and hyperpolarizes K⁺ channels. The activation of 5-HT5ARs activate the formation of cyclic ADP ribose (cADPR), and in turn elicit Ca²⁺ release. However, 5-HT4, 5-HT6, and 5-HT7 receptors stimulate AC, resulting in increased cAMP level, activated protein kinase A (PKA), and the regulation of several signaling molecules, such as CREB. The MAPK pathway concludes with the activation of Erk1 and Erk2 proteins which in turn enter the nucleus and cause phosphorylation-mediated activation of transcription factors, such as Elk1, and CREB. When 5-HT binds to 5-HT3Rs, a gate opens up and becomes permeable to Na⁺, K⁺, and Ca²⁺ ions. Ca²⁺ decreases 5-HT-induced Na⁺ currents in a concentration-dependent manner without affecting the internal alteration of the current/voltage relation (Adayev et al. 2005; Filip and Bader 2009; Engel et al. 2013; Locher et al. 2017)
Serotonergic Pathways. In addition to the dorsal and median raphe nuclear complex, many serotonergic neurons are found in the reticular region of the lower brain stem, which is subdivided into caudal and rostral groups. Serotonergic neurons of the rostral system (caudal linear, dorsal raphe, and median raphe nuclei) mostly terminate in the dorsal and medial raphe nuclei, which in turn innervate much of the rest of the CNS by diffuse projections. Serotonergic neurons of the caudal group (raphe magnus, raphe obscurus, raphe pallidus nuclei) descend into the brain stem and the spinal cord along the dorsal horn, ventral horn motor nuclei and thoracic cord intermediolateral column and are mostly involved in sensory, motor and autonomic functioning. The central gray matter and the ependyma of the central canal also receives 5-HT input in a pattern of 5-HT nerve plexus. Serotonergic signaling regulates impulsivity and behavioral adaptation through actions within the medial prefrontal cortex (mPFC). Serotonergic innervation is present throughout the NAc and involved in social behaviors. Moreover, serotonergic fibers innervate the amygdala and enhance aversive memory acquisition. Signaling of 5-HT in the periaqueductal gray (PAG) is involved in behavioral inhibition and prevent panic-like behaviors. Additionally, serotonergic innervation is present throughout the bed nucleus of the stria terminalis (BNST) and could promote or inhibit approach and avoidance behaviors. The expression of the fear- and anxiety-related defensive behavioral responses of the habenula (Hb) is also regulated by serotonergic projections. The presence of 5-HT has been observed in the pons, medulla, septal area and specific areas of the thalamus, hypothalamus, substantia nigra (SN) and locus coeruleus (LC). The hippocampus (Hippo), the suprachiasmatic nucleus, the olfactory bulb and the medial septum nucleus receive 5-HT innervation from the MRN. Likewise, serotonergic fibers are one of the afferent fibers innervating and affecting the activity of cerebellar circuitry (Dölen 2015; Jorgensen 2007; Puig and Gulledge 2011; Linley et al. 2017; Saitow et al. 2012)
A summary of the major nervous system diseases associated with dysfunctions in serotonergic receptors
The serotonergic system extends throughout the central nervous system (CNS) and the gastrointestinal (GI) tract. In the CNS, serotonin (5-HT, 5-hydroxytryptamine) modulates a broad spectrum of functions, including mood, cognition, anxiety, learning, memory, reward processing, and sleep. These processes are mediated through 5-HT binding to 5-HT receptors (5-HTRs), are classified into seven distinct groups. Deficits in the serotonergic system can result in various pathological conditions, particularly depression, schizophrenia, mood disorders, and autism. In this review, we outlined the complexity of serotonergic modulation of physiologic and pathologic processes. Moreover, we provided experimental and clinical evidence of 5-HT's involvement in neuropsychiatric disorders and discussed the molecular mechanisms that underlie these illnesses and contribute to the new therapies.
 
TTC3 and cognitive impairment The involvement of TTC3 in cognitive impairment may rely on PQC mechanisms. TTC3 functions as both a molecular cochaperone, ubiquitin E3 ligase, and major regulator of mitochondrial function
Structure and composition of TTC3 Four TPR motifs of TTC3 are located at 231–264 aa, 266–298 aa, 536–572 aa, and 576–609 aa. The RING finger domain is located at 1957–1997 aa. Ser378 is a possible Akt phosphorylation site. NLS is only known to be located in the 1–650 aa range
Role of TTC3 in cognitive impairment TTC3 is involved in the proliferation and differentiation of nerve cells through the regulation of the RhoA, ROCK, CIT-N, PIIa, and Akt signaling pathways and affects mitochondrial function by POLG ubiquitination, which may eventually lead to cognitive impairment
TTC3-mediated PQC mechanism a TTC3 acts as a cochaperone involved in the ubiquitin-protease degradation process and enters the nucleus to form aggregates mediated by the NLS, with the N-terminus playing a key role. b TTC3 is involved in the ubiquitination process as an E3 ubiquitin ligase, and the RING finger domain is the critical element of TTC3 function. c TTC3 affects mitochondrial function by regulating POLG, which promotes autophagy and ultimately neuronal loss
The tetrapeptide repeat domain 3 (TTC3) gene falls within Down's syndrome (DS) critical region. Cognitive impairment is a common phenotype of DS and Alzheimer’s disease (AD), and overexpression of TTC3 can accelerate cognitive decline, but the specific mechanism is unknown. The TTC3-mediated protein quality control (PQC) mechanism, similar to the PQC system, is divided into three parts: it acts as a cochaperone to assist proteins in folding correctly; it acts as an E3 ubiquitin ligase (E3s) involved in protein degradation processes through the ubiquitin–proteasome system (UPS); and it may also eventually cause autophagy by affecting mitochondrial function. Thus, this article reviews the research progress on the structure, function, and metabolism of TTC3, including the recent research progress on TTC3 in DS and AD; the role of TTC3 in cognitive impairment through PQC in combination with the abovementioned attributes of TTC3; and the potential targets of TTC3 in the treatment of such diseases.
 
Antibodies and oxidative stress are hallmarks of multiple sclerosis (MS) lesions. We aimed to clarify the relation between them, their role in MS patients and to investigate their specificity, comparing MS with classical neurodegenerative diseases (ND). Brain samples from 14 MS cases, 6 with ND and 9 controls (without neurological diseases). Immunohistochemistry assays were used to detect oxidized lipids (EO6), IgG and IgM, oligodendrocytes (Olig2), axons (NF, neurofilament) and cellular (TUNEL) and axonal damage (APP, amyloid precursor protein). We did not observe EO6 in controls. All samples from MS patients showed EO6 in oligodendrocytes and axons within lesions. We did not detect co-localization between EO6 and antibodies. Neither did we between EO6 and TUNEL or APP. 94.4% of TUNEL-positive cells in normal appearing white matter were also stained for IgG and 75.5% for IgM. IgM, but not IgG, co-localized with APP. EO6 was associated with axonal damage in amyotrophic lateral sclerosis (ALS). We did not observe association between antibodies and cellular or axonal damage in ND patients. MS patients showed a higher number of B cells and plasma cells in the lesions and meninges than controls. The number of B cells and plasma cells was associated with the presence of antibodies and with the activity of the lesions. We observed a main role of B lymphocytes in the development of MS lesions. Antibodies contribute to the oligodendrocyte and axonal damage in MS. Oxidative stress was associated with axonal damage in ALS.
 
Tumor Necrosis Factor (TNF)-α is a proinflammatory cytokine (PIC) and has been implicated in a variety of illness including cardiovascular disease. The current study investigated the inflammatory response trigged by TNFα in both cultured brain neurons and the hypothalamic paraventricular nucleus (PVN), a key cardiovascular relevant brain area, of the Sprague Dawley (SD) rats. Our results demonstrated that TNFα treatment induces a dose- and time-dependent increase in mRNA expression of PICs including Interleukin (IL)-1β and Interleukin-6 (IL6); chemokines including C–C Motif Chemokine Ligand 5 (CCL5) and C–C Motif Chemokine Ligand 12 (CCL12), inducible nitric oxide synthase (iNOS), as well as transcription factor NF-kB in cultured brain neurons from neonatal SD rats. Consistent with this finding, immunostaining shows that TNFα treatment increases immunoreactivity of IL1β, CCL5, iNOS and stimulates activation or expression of NF-kB, in both cultured brain neurons and the PVN of adult SD rats. We further compared mRNA expression of the aforementioned genes in basal level as well as in response to TNFα challenge between SD rats and Dahl Salt-sensitive (Dahl-S) rats, an animal model of salt-sensitive hypertension. Dahl-S brain neurons presented higher baseline levels as well as greater response to TNFα challenge in mRNA expression of CCL5, iNOS and IL1β. Furthermore, central administration of TNFα caused significant higher response in CCL12 in the PVN of Dahl-S rats. The increased inflammatory response to TNFα in Dahl-S rats may be indicative of an underlying mechanism for enhanced pressor reactivity to salt intake in the Dahl-S rat model.
 
a Bioinformatics analysis for prediction of rs10773771 influence on RNA folding structures; MFE minimum free energy. b Association between rs10773771 genotypes and PIWIL1 expression in human skin tissues based on GTEx V7. c Association between rs10773771 genotypes and PIWIL1 expression in human thyroid tissues based on GTEx V7. d Allele frequency of rs10773771 among different populations from the 1000 genome project. Population descriptions: AMR American, AFR African, EUR European, EAS East Asian, SAS South Asian
Convincing evidence has shown that microRNAs (miRNAs) are involved in the pathogenesis of stroke. This study aimed to examine whether miRNA biogenesis genes polymorphisms are associated with risk of large artery atherosclerosis (LAA) stroke. Three polymorphisms (DROSHA rs10719 T>C, RAN rs3803012 A>G, and PIWIL1 rs10773771 C>T) were screened by certain criteria. A total of 1,785 (710 cases and 1,075 controls) study subjects were included in this study. We found that rs10773771 CC genotype was associated with a decreased risk of LAA stroke (CC vs. TT/CT: OR 0.63, 95% CI 0.46–0.86, P = 3 × 10–3). In silico analysis suggested that rs10773771 can change the mRNA secondary structure of PIWIL1 and affect the binding of the miRNAs and regulatory motifs to the 3′-UTR of PIWIL1. Expression quantitative trait loci analysis showed that rs10773771 could change the expression of PIWIL1 in human skin (P = 1.534 × 10–10) and thyroid tissues (P = 4.869 × 10–6). These findings suggested that PIWIL1 rs10773771 may be associated with a decreased risk of LAA stroke.
 
With the increase in fetal surgeries, the effect of maternal anesthesia on progeny has attracted much attention. Our previous studies have demonstrated that 3.5% sevoflurane maternal exposure resulted in over-activated autophagy and cognitive impairment in the offspring. The autophagy activation resulted in increased apoptosis and decreased proliferation. However, the effects of sevoflurane on neural stem cell (NSC) differentiation is unclear. There is evidence that autophagy might participate in anesthesia-induced NSC differentiation. Firstly, we examined the effects of sevoflurane on NSC differentiation and explored possible mechanisms. Then, we investigated whether autophagy was related to differentiation. On gestational day 14 (G14), rats were exposed to 2% or 3.5% sevoflurane for 2 h, then markers of neurons and astrocytes, and the FOXO3 expression was measured in fetal brains 48 h later. The differentiation of NSCs was detected after autophagy inhibition by 3-MA. Changes in NSC differentiation, autophagy level, and FOXO3 were examined after administration of lithium chloride. After 3.5% sevoflurane exposure, the expressions of β-Tubulin III, NeuN, SYP, GFAP and FOXO3 increased. Autophagy inhibition alleviates improper NSC differentiation. Lithium chloride attenuated FOXO3 and autophagy activation, ameliorated NSC differentiation and the decline of Nestin expression. Our results demonstrated that maternal exposure to 3.5% sevoflurane for 2 h during the mid-trimester induced NSC differentiation in the fetal brain through the activation of FOXO3. Autophagy inhibitor or lithium chloride reversed the improper differentiation of NSCs.
 
Subcellular localization, expression, and function of rat REV-ERBβ in undifferentiated and neuronally differentiated cultured rat AHPs. a Cultured rat AHPs in the undifferentiated maintenance state were analyzed by confocal laser fluorescence microscopy using anti-REV-ERBβ, anti-Nestin, and anti-Ki67 antibodies. Merged images show REV-ERBβ (white), Nestin (red), Ki67 (green), and nuclear staining with DAPI (blue). Scale bar 20 µm. b Rat AHPs, 7 days after the induction of neural differentiation, were immunostained and analyzed by fluorescence microscopy. Images of fluorescence staining with an anti-REV-ERBβ antibody (green), anti-βIII tubulin antibody (red), and DAPI (blue) are shown. Scale bar 50 µm. c Comparison of rat Rev-erbβ expression levels between undifferentiated and neuronally differentiated cultured rat AHPs. The gene expression levels were compared by quantitative RT-PCR in rat AHPs cultured in the undifferentiated state (Undiff.) and rat AHPs at 7 days after induction of neural differentiation (Neural diff.). Data are expressed as the mean ± SD (****P < 0.0001). Statistical comparisons were performed with a two-tailed unpaired t test followed by Welch’s correction. d Function of rat Rev-erbβ in undifferentiated and neuronally differentiated cultured rat AHPs. Rat AHPs cultured in an undifferentiated maintenance state were transfected with gene knockdown vector plasmid DNA expressing scrambled shRNA sequences and shRNA sequences targeting rat Rev-erbβ. After 48 h, proliferating cells were labeled by BrdU uptake. GFP is expressed under the control of the PGK promoter in transfected cells. The graph represents the percentage of cells transfected with the scrambled (CTRL, white bar) and Rev-erbβ (Revβ-KD, black bar) shRNA vectors that were double positive for BrdU and GFP. Data are presented as the mean ± SEM (****P < 0.0001). Statistical comparisons were performed with a two-tailed unpaired t test followed by Welch’s correction. e Neural differentiation was induced immediately after gene knockdown. The neurite lengths of 18 differentiated cells were measured 7 days after induction of neural differentiation following transfection with vector DNA expressing scrambled (CTRL, white bar) or Rev-erbβ (Revβ-KD, gray bar) shRNA. Neurite lengths were analyzed and plotted (CTRL, white circles; Revβ-KD, triangles). Data are presented as the median and interquartile range (***P < 0.001). Statistical comparisons were performed by a two-tailed unpaired t test followed by Welch’s correction
Relationships between REV-ERB agonist SR9009 concentrations and the proliferation of cultured rat AHPs. a SR9009 (0–5 µM) was added to rat AHPs cultured in undifferentiated maintenance medium for 24 h, and the cells were labeled with BrdU. BrdU-positive cells were detected and measured using an anti-BrdU antibody. Data in the graph are expressed as the mean ± SEM (*P < 0.05, ****P < 0.0001). Statistical comparisons were performed using Dunnett’s multiple comparisons test after ANOVA. Gene knockdown vector plasmids expressing GFP and scrambled (b) or Rev-erbβ-targeted (c) shRNA sequences were introduced into rat AHPs cultured in an undifferentiated maintenance state. Vehicle or SR9009 (2.5 µM) solutions was added to the medium, and cells were cultured for an additional 24 h. Proliferating cells were labeled with BrdU, and BrdU-positive cells were detected and measured using an anti-BrdU antibody. Data (CTRL, white bars; Revβ-KD, black bars) in the graphs are expressed as the mean ± SEM (***P < 0.001). Statistical comparisons were performed by two-tailed unpaired t tests followed by Welch’s correction
Effects of the REV-ERB agonist SR9009 on neurogenesis and neurite outgrowth and suppression in rat AHPs via the REV-ERBβ-dependent pathway. a SR9009 regulates neurite outgrowth in a concentration-dependent manner. Rat AHPs were cultured for 7 days in neural differentiation medium containing SR9009 (0–5 µM), which was added immediately after the induction of neural differentiation. Neurons and neurites were detected with an antibody against βIII-tubulin, a marker of neuronal differentiation, and neurite lengths were measured. Data in the graph are expressed as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Statistical comparisons were performed using Dunnett’s multiple comparisons test after ANOVA. Gene knockdown vector plasmids expressing GFP and scrambled (b; white bars) or Rev-erbβ-targeted (c; gray bars) shRNA sequences were transfected into cultured rat AHPs, followed by 7 days of culture in neural differentiation medium containing low (0.1 µM; b, c) or high (2.5 µM; d, e) concentrations of SR9009. Neuronal projections from GFP-positive cells were detected by immunostaining with an anti-βIII tubulin antibody, and their lengths were measured. The neurite lengths of 18 differentiated cells were analyzed and plotted on a graph (Vehicle, white circles; SR9009 addition, triangles). Data are expressed as the median and interquartile range (**P < 0.01). Statistical comparisons were performed by two-tailed unpaired t tests followed by Welch’s correction
Changes in the expression levels of Rev-erbβ and REV-ERBβ target genes following treatment with the REV-ERBβ agonist SR9009. Rat AHPs in the undifferentiated maintenance state were treated with Vehicle (white bars) or high-concentration (2.5 µM) SR9009 (black bars) for 24 h, and the expression levels of Rev-erbβ (a) and Ccna2 (b) were examined by quantitative RT-PCR. Data are expressed as the mean ± SD (****P < 0.0001). Statistical comparisons were performed by two-tailed unpaired t tests followed by Welch’s correction. Vehicle (white bar), low- (0.1 µM), or high-concentration (2.5 µM) SR9009 (gray bars) was added to the neural differentiation-inducing culture of rat AHPs for 7 days. Quantitative RT-PCR was performed on the collected cell samples, and the expression levels of Rev-erbβ (c) and Sez6 (d) were evaluated in each group. Data are expressed as the mean ± SD (****P < 0.0001). Statistical comparisons were performed by Dunnett’s multiple comparisons after ANOVA
of the study findings. Right panel: SR9009 repressively regulates the cell proliferation machinery of cultured rat AHPs via REV-ERBβ or other pathways. The gene expression of Cyclin A2 (Ccna2), a representative example of a REV-ERBβ target gene, is repressed. Left panel: SR9009 enhances neurite outgrowth during the neurogenesis of cultured rat AHPs at 0.1 µM and acts in an inhibitory manner at 2.5 µM. REV-ERBβ target genes, represented by Seizure-related gene 6 (Sez6), are up- and down regulated in an SR9009 concentration-dependent manner. Red arrow: activation, black lines: repression
REV-ERBs are heme-binding nuclear receptors that regulate the circadian rhythm and play important roles in the regulation of proliferation and the neuronal differentiation process in neuronal stem/progenitor cells in the adult brain. However, the effects of REV-ERB activation in the adult brain remain unclear. In this study, SR9009, a synthetic REV-ERB agonist that produces anxiolytic effects in mice, was used to treat undifferentiated and neuronally differentiated cultured rat adult hippocampal neural stem/progenitor cells (AHPs). The expression of Rev-erbβ was upregulated during neurogenesis in cultured rat AHPs, and Rev-erbβ knockdown analysis indicated that REV-ERBβ regulates the proliferation and neurite outgrowth of cultured rat AHPs. The application of a low concentration (0.1 µM) of the REV-ERB agonist SR9009 enhanced neurite outgrowth during neurogenesis in cultured rat AHPs, whereas the addition of a high concentration (2.5 µM) of SR9009 suppressed neurite outgrowth. Further examination of the SR9009 regulatory mechanism showed that the expressions of downstream target genes of REV-ERBβ, including Ccna2 and Sez6, were modulated by SR9009. The results of this study indicated that REV-ERBβ activity in cultured rat AHPs was regulated by SR9009 in a concentration-dependent manner. Furthermore, SR9009 inhibited the growth of cultured rat AHPs through various pathways, which may provide insight into the multifunctional mechanisms of action associated with SR9009. The findings of this study may provide an improved understanding of proliferation and neuronal maturation mechanisms in cultured rat AHPs through SR9009-regulated REV-ERBβ signaling pathways.
 
SHANK family gene structure, protein domains and isoforms. Panels a, b and c show the chromosome locations, gene structures, domains and isoforms of SHANK1, SHANK2 and SHANK3, respectively. The protein-coding domains are marked and aligned to corresponding exons, including ANK, PDZ, PRO, SH3 and SAM. The positions of two promoters are indicated by black arrows. The exons in SHANK2 in red are alternatively spliced, with three identifiable promoters indicated by black arrows and two alternative stop codons (TAA) pointed by yellow arrows (b). The exons of SHANK3 in red are also alternatively spliced, with exon 11a being newly identified. The positions of six identified promoters are indicated by black arrows (c). ANK ankyrin repeat domain, PDZ postsynaptic density protein 95-discs large homologue 1-zonula occludens 1 domain, PRO proline-rich region, SAM sterile alpha motif, SH3 SRC homology 3 domain superfamily
Schematic drawing illustrating the potential protein–protein interaction network of the SHANK at the postsynaptic density of glutamatergic synapses. NMDA-R N-methyl-d-aspartate receptor, BDNF brain-derived neurotrophic factor, AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, DLGAP1, 2, 3 discs large associated protein 1, 2, 3, SLC6A3 solute carrier family 6 (neurotransmitter transporter, dopamine), member 3
The Shank family proteins are enriched at the postsynaptic density (PSD) of excitatory glutamatergic synapses. They serve as synaptic scaffolding proteins and appear to play a critical role in the formation, maintenance and functioning of synapse. Increasing evidence from genetic association and animal model studies indicates a connection of SHANK genes defects with the development of neuropsychiatric disorders. In this review, we first update the current understanding of the SHANK family genes and their encoded protein products. We then denote the literature relating their alterations to the risk of neuropsychiatric diseases. We further review evidence from animal models that provided molecular insights into the biological as well as pathogenic roles of Shank proteins in synapses, and the potential relationship to the development of abnormal neurobehavioral phenotypes.
 
Alzheimer's disease (AD) is the most common age-associated dementia with complex pathological hallmarks. Mitochondrion, synaptosome, and myelin sheath appear to be vulnerable and play a key role in the pathogenesis of AD. To clarify the early mechanism associated with AD, followed by subcellular components separation, we performed iTRAQ (isobaric tags for relative and absolute quantification)-based proteomics analysis to simultaneously investigate the differentially expressed proteins (DEPs) within the mitochondria, synaptosome, and myelin sheath in the cerebrum of the 6-month-old triple transgenic AD (3 × Tg-AD) and 6-month-old wild-type (WT) mice. A large number of DEPs between the AD and WT mice were identified. Most of them are related to mitochondria and synaptic dysfunction and cytoskeletal protein change. Differential expressions of Lrpprc, Nefl, and Sirpa were verified by Western blot analysis. The results suggest that decreased energy metabolism, impaired amino acid metabolism and neurotransmitter synthesis, increase compensatory fatty acid metabolism, up-regulated cytoskeletal protein expression, and oxidative stress are the early events of AD. Among these, mitochondrial damage, synaptic dysfunction, decreased energy metabolism, and abnormal amino acid metabolism are the most significant events. The results indicate that it is feasible to separate and simultaneously perform proteomics analysis on the three subcellular components.
 
Top-cited authors
Grazyna Lietzau
  • Medical University of Gdansk
Monika Waśkow
  • Akademia Pomorska w Slupsku
Przemyslaw Kowianski
  • Medical University of Gdansk
Ewelina Czuba
  • Medical University of Gdansk
Janusz Morys
  • Pomeranian Medical University in Szczecin