Molecular Neurobiology

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  • Chen ShenChen Shen
  • Cong ChenCong Chen
  • Tong WangTong Wang
  • [...]
  • Wei-Ping ZhangWei-Ping Zhang
Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in the salvaging synthesis pathway of the nicotinamide adenine dinucleotide (NAD). Both NAMPT and NAD progressively decline upon aging and neurodegenerative diseases. The depletion of NAMPT induces mitochondrial dysfunction in motor neurons and causes bioenergetic stress in neurons. However, the roles of NAMPT in hippocampus neurons need to be further studied. Using floxed Nampt (Namptflox/flox) mice, we knocked out Nampt specifically in the hippocampus CA1 neurons by injecting rAAV-hSyn-Cre-APRE-pA. The depletion of NAMPT in hippocampus neurons induced cognitive deficiency in mice. Nevertheless, no morphological change of hippocampus neurons was observed with immunofluorescent imaging. Under the transmission electron microscope, we observed mitochondrial swollen and mitochondrial number decreasing in the cell body and the neurites of hippocampus neurons. In addition, we found the intracellular Aβ (6E10) increased in the hippocampus CA1 region. The intensity of Aβ42 remained unchanged, but it tended to aggregate. The GFAP level, an astrocyte marker, and the Iba1 level, a microglia marker, significantly increased in the mouse hippocampus. In the primary cultured rat neurons, NAMPT inhibition by FK866 decreased the NAD level of neurons at > 10⁻⁹ M. FK866 dropped the mitochondrial membrane potential in the cell body of neurons at > 10⁻⁹ M and in the dendrite of neurons at > 10⁻⁸ M. FK866 decreased the number and shortened the length of branches of neurons at > 10⁻⁷ M. Together, likely due to the injury of mitochondria, the decline of NAMPT level can be a critical risk factor for neurodegeneration.
 
  • Pierluigi ValentePierluigi Valente
  • Antonella MarteAntonella Marte
  • Francesca FranchiFrancesca Franchi
  • [...]
  • Fabio BenfenatiFabio Benfenati
Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na ⁺ channels Na V 1.2/Na V 1.6 predominantly expressed in brain glutamatergic neurons. Na V channels form complexes with β-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and β-subunits on Na V channels raises the question of whether PRRT2 and β-subunits interact or compete for common binding sites on the α-subunit, generating Na ⁺ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that β-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of Na V 1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the β4-induced increase in expression and functional activation of the transient and persistent Na V 1.2 currents, without affecting resurgent current. The data indicate that β4-subunit and PRRT2 form a push–pull system that finely tunes the membrane expression and function of Na V channels and the intrinsic neuronal excitability.
 
  • Jie ShaoJie Shao
  • Xiang YinXiang Yin
  • Yue LangYue Lang
  • [...]
  • Li CuiLi Cui
Modulation of microglial pro/anti-inflammatory states and autophagy are promising new therapies for ischemic stroke, but the underlying mechanisms remain largely unexplored. The objective of the study is to determine the intrinsic role of PrPC (cellular prion protein) in the regulation of microglial inflammatory states and autophagy in ischemic stroke. PrPC was expressed in murine microglia, and an in vitro oxygen–glucose deprivation/reperfusion (OGD/R) model was established in microglia of different PRNP genotypes. During reperfusion following OGD, wild-type (WT) microglia had significantly increased pro/anti-inflammatory microglial percentages and related cytokine [interleukin [IL]-6, IL-10, IL-4, tumor necrosis factor, and interferon-gamma] release at reperfusion after 48 or 72 h. WT microglia also showed greater accumulation of the autophagy markers LC3B-II/I (microtubule-associated protein B-light chain 3), but not of p62 or LAMP1 (lysosome-associated membrane protein) at reperfusion after 24 h and 48 h. Inhibition of autophagy using 3-methyladenine or bafilomycin A1 aggravated the OGD/R-induced pro-inflammatory state, and the effect of 3-methyladenine was significantly stronger than that of bafilomycin A1. Concomitantly, PRNP knockout shortened the accumulation of LC3B-II/I, suppressed microglial anti-inflammatory states, and further aggravated the pro-inflammatory states. Conversely, PRNP overexpression had the opposite effects. Bafilomycin A1 reversed the effect of PrPC on microglial inflammatory state transformation. Moreover, microglia with PRNP overexpression exhibited higher levels of LAMP1 expression in the control and OGD/R groups and delayed the OGD/R-induced decrease of LAMP1 to reperfusion after 48 h. PrPC attenuates OGD/R-induced damage by skewing microglia toward an anti-inflammatory state via enhanced and prolonged activation of autophagy. Graphical Abstract
 
  • Ximeng YangXimeng Yang
  • Chihiro TohdaChihiro Tohda
Galectin-1 (Gal-1), a member of the Galectin family, is expressed in various tissues and responsible for multiple biological activities. Previous studies reported that extracellular Gal-1 participated in axonal growth and repair, and Gal-1 knockout mice exhibited memory impairment. However, no study has demonstrated the direct contribution of intracellular Gal-1 upregulation in neurons to promoting axonal regeneration in the brain and recovering memory function. In the present study, we found that axonal growth is promoted by overexpression of Gal-1 via adeno-associated virus serotype 9 delivery in primary cultured hippocampal neurons. Moreover, Gal-1 was expressed on the membranes of growth cones in hippocampal neurons and interacted with a novel axonal guidance molecule, Secernin-1, which was secreted from prefrontal cortex (PFC) neurons. Gal-1-overexpression-driven axonal growth was enhanced when recombinant (extracellular) Secernin-1 was treated to the axonal site in a neuron device chamber. Direct binding of extracellular Secernin-1 with Gal-1 was detected through immunoprecipitation and immunocytochemistry, demonstrating that Gal-1 possibly works as an axonal guidance receptor for Secernin-1 in hippocampal neurons. In the PFC, the expression of Gal-1 in axonal shafts and terminals of hippocampal neurons was decreased in the 5XFAD mouse model of Alzheimer’s disease (AD). Overexpression of Gal-1 in hippocampal neurons recovered memory deficits and induced axonal regeneration toward the PFC in 5XFAD mice. This study suggests that the enhanced interaction of Secernin-1 and Gal-1 can be harnessed as a therapeutic strategy for long-distance and direction-specific axonal regeneration in AD.
 
The molecular structure of TRPV4 and its major sites for channel activation and sensitization. TRPV4 has six transmembrane alpha-helices (S1–S6) with its NH2 and COOH terminus localizing in the cytoplasm. The pore that allows the ionic flow is located between S5 and S6 domains. Its NH2 terminus plays a vital role in mediating responses to heat, shear stress, and hypotonic cell swelling. Its COOH terminus maintains channel protein folding, maturation, and trafficking
TRPV4 plays an important role in neuronal apoptosis and neurotoxicity during epilepsy and ischemia. TRPV4 activation leads to the activation of microglia and astrocytes, and the increase of NLRP3 inflammasome expression to produce more proinflammatory cytokines (IL-6, IL-1β, TNF-α, and IL-18), which is involved in the neuronal injury in epilepsy (①). During epilepsy and ischemia, the expression and activation of TRPV4 are increased. Activation of TRPV4 induces neuronal apoptosis via downregulating PI3K/Akt and upregulating P38 MAPK signaling pathways (②, ③). Activation of TRPV4 promotes presynaptic glutamate release, increases the postsynaptic AMPA and NMDA receptor function, and inhibits postsynaptic GABA receptor function, through activating PKA/PKC signal pathway (④), upregulating P38 MAPK (②), Ca²⁺/CaMKII (⑤, ⑥) and NR2B-NMDAR signal pathways (⑤), and downregulating PI3K/Akt signal pathway (③), thereby disrupting the balance between the excitatory and inhibitory neurotransmitter systems, increasing neuronal excitability, and ultimately leading to excitatory toxicity
The role of TRPV4 in tumor onset and progression and its potential mechanism. TRPV4 affects the proliferation, differentiation, apoptosis, migration, invasion, and metastasis of tumor cells through different pathways
Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel that can be activated by diverse stimuli, such as heat, mechanical force, hypo-osmolarity, and arachidonic acid metabolites. TRPV4 is widely expressed in the central nervous system (CNS) and participates in many significant physiological processes. However, accumulative evidence has suggested that deficiency, abnormal expression or distribution, and overactivation of TRPV4 are involved in pathological processes of multiple neurological diseases. Here, we review the latest studies concerning the known features of this channel, including its expression, structure, and its physiological and pathological roles in the CNS, proposing an emerging therapeutic strategy for CNS diseases.
 
In the present study, the effect of 6-((4-fluorophenyl) selanyl)-9H-purine (FSP) was tested against memory impairment and sensitivity to nociception induced by intracerebroventricular injection of amyloid-beta peptide (Aβ) (25–35 fragment), 3 nmol/3 μl/per site in mice. Memory impairment was determined by the object recognition task (ORT) and nociception by the Von-Frey test (VFT). Aβ caused neuroinflammation with upregulation of glial fibrillary acidic protein (GFAP) (in hippocampus), nuclear factor-κB (NF-κB), and the proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in cerebral cortex and hippocampus. Additionally, Aβ increased oxidant levels and lipid peroxidation in cerebral cortex and hippocampus, but decreased heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prdx1) expression in the hippocampus. Anti-neuroinflammatory effects of FSP were demonstrated by a decrease in the expression of GFAP and NF-κB in the hippocampus, as well as a decrease in proinflammatory cytokines in both the hippocampus and cerebral cortex FSP protected against oxidative stress by decreasing oxidant levels and lipid peroxidation and by increasing HO-1 and Prdx1 expressions in the hippocampus of mice. Moreover, FSP prevented the activation of nuclear factor erythroid 2-related factor 2 (Nrf-2) in the hippocampus of mice induced by Aβ. In conclusion, treatment with FSP attenuated memory impairment, nociception sensitivity by decreasing oxidative stress, and neuroinflammation in a mouse model of Alzheimer’s disease. Graphical Abstract
 
Human molecular network analysis of CASP1, CASP4, and CASP5 using String. A network analysis was performed with human CASP1, CASP4, and CASP5 using String (© String Consortium 2022). A minimum required interaction score of > 0.900 (very high confidence) was the criterion used to determine protein interaction, which is a culmination of evidence from known, predicted, and other interactions, and represented by different coloured edges
Expression changes in blood samples from neuropathic pain patients versus non-neuropathic pain controls with FDR correction and controlling for age and gender. Normalised relative quantity (NRQ) of differential expression in controls and neuropathic pain (NP) patients for all genes declared significant under any rubric. Horizontal lines through grouped data denote mean values and standard deviations
ROC curves comparing groups. ROC curves for A neuropathic versus nociceptive pain (< 12 S-LANSS score), disregarding controls for gene combination 17 – PLAC8, ROMO1, and A3GALT2. B–F neuropathic pain versus controls, disregarding nociceptive pain (< 12 S-LANSS score) for respective gene combinations 7 – CASP4, CASP5, CASP9; 10 – CASP5, CASP9, TMEM88; 12 – CASP5, CASP9, FRP2; 15 – CASP9, FPR2, TMEM88; 19 – CASP4, CCR5, SH3BGRL3; and 23 – CASP9, SH3BGRL3, TMEM88
Steps to identify and develop biomarkers for clinical use. The first step in biomarker discovery includes biomarker identification and assay development. Biomarkers can either be identified in preclinical models and reverse-translated to humans or vice versa. The next step is biomarker development, which includes determining that the biomarker or analyte is easily measurable, and that the detection method is reliable and reproducible. A reproducible, reliable, sensitive, and specific biomarker or combination of biomarkers will then be in a position for clinical use. The figure has been adopted from Davis et al. (2020) [59] and permissions have been acquired from the Springer Nature Copyright Clearance Centre
Neuropathic pain is a common chronic condition, which remains poorly understood. Many patients receiving treatment continue to experience severe pain, due to limited diagnostic/treatment management programmes. The development of objective clinical diagnostic/treatment strategies requires identification of robust biomarkers of neuropathic pain. To this end, we looked to identify biomarkers of chronic neuropathic pain by assessing gene expression profiles in an animal model of neuropathic pain, and differential gene expression in patients to determine the potential translatability. We demonstrated cross-species validation of several genes including those identified through bioinformatic analysis by assessing their expression in blood samples from neuropathic pain patients, according to conservative assessments of significance measured using Bonferroni-corrected p-values. These include CASP5 (p = 0.00226), CASP8 (p = 0.00587), CASP9 (p = 2.09 × 10⁻⁹), FPR2 (p = 0.00278), SH3BGRL3 (p = 0.00633), and TMEM88 (p = 0.00038). A ROC analysis revealed several combinations of genes to show high levels of discriminatory power in the comparison of neuropathic pain patients and control participants, of which the combination SH3BGRL3, TMEM88, and CASP9 achieved the highest level (AUROC = 0.923). The CASP9 gene was found to be common in five combinations of three genes revealing the highest levels of discriminatory power. In contrast, the gene combination PLAC8, ROMO1, and A3GALT2 showed the highest levels of discriminatory power in the comparison of neuropathic pain and nociceptive pain (AUROC = 0.919), when patients were grouped by S-LANSS scores. Molecules that demonstrate an active role in neuropathic pain have the potential to be developed into a biological measure for objective diagnostic tests, or as novel drug targets for improved pain management.
 
Despite the extensive use of the cuprizone (CPZ) demyelination animal model, there is little evidence regarding the effects of CPZ on a cellular level. Initial studies have suggested that oligodendrocytes (OL) are the main cell targets for CPZ toxicity. However, recent data have revealed additional effects on neural stem cells and progenitor cells (NSC/NPC), which constitute a reservoir for OL regeneration during brain remyelination. We cultured NSC/NPC as neurospheres to investigate CPZ effects on cell mechanisms which are thought to be involved in demyelination and remyelination processes in vivo. Proliferating NSC/NPC cultures exposed to CPZ showed overproduction of intracellular reactive oxygen species and increased progenitor migration at the expense of a significant inhibition of cell proliferation. Although NSC/NPC survival was not affected by CPZ in proliferative conditions, we found that CPZ-treated cultures undergoing cell differentiation were more prone to cell death than controls. The commitment and cell differentiation towards neural lineages did not seem to be affected by CPZ, as shown by the conserved proportions of OL, astrocytes, and neurons. Nevertheless, when CPZ treatment was performed after cell differentiation, we detected a significant reduction in the number and the morphological complexity of OL, astrogliosis, and neuronal damage. We conclude that, in addition to damaging mature OL, CPZ also reduces NSC/NPC proliferation and activates progenitor migration. These results shed light on CPZ direct effects on NSC proliferation and the progression of in vitro differentiation.
 
Inflammation has been associated with numerous neurological disorders. Inflammatory environments trigger a series of cellular and physiological alterations in the brain. However, how inflammatory milieu affects neuronal physiology and how neuronal alterations progress in the inflammatory environments are not fully understood. In this study, we examined the effects of pro-inflammatory milieu on mitochondrial functions and neuronal activities in the hypothalamic POMC neurons. Treating mHypoA-POMC/GFP1 with the conditioned medium collected from LPS activated macrophage were employed to mimic the inflammatory milieu during hypothalamic inflammation. After a 24-h treatment, intracellular ROS/RNS levels were elevated, and the antioxidant enzymes were reduced. Mitochondrial respiration and mitochondrial functions, including basal respiratory rate, spared respiration capacity, and maximal respiration, were all significantly compromised by inflammatory milieu. Moreover, pro-inflammatory cytokines altered mitochondrial dynamics in a time-dependent manner, resulting in the elongation of mitochondria in POMC neurons after a 24-h treatment. Additionally, the increase of C-Fos and Pomc genes expression indicated that the neurons were activated upon the stimulation of inflammatory environment. This neuronal activation of were confirmed on the LPS-challenged mice. Collectively, a short-term to midterm exposure to inflammatory milieu stimulated metabolic switch and neuronal activation, whereas chronic exposure triggered the elevation of oxidative stress, the decrease of the mitochondrial respiration, and the alterations of mitochondrial dynamics.
 
Transducer design, characterization of transducer for rat skull. A Stereotaxic images of the nucleus accumbens (NAc) in the coronal sections of the rat brain (the picture is taken and adapted from “The Rat Brain in Stereotaxic Coordinates,7. Print, Paxinos and Watson 2013”). B 3 dB (acoustic intensity is within 50% of the peak) beam patterns for (a) 3 mm aperture at 2.4 MHz, (b) 6 mm aperture at 2.4 MHz, (c) 3 mm aperture at 5 MHz, and (d) 6 mm aperture at 5 MHz. C Ultrasonic transmission image of a rat skull at 6.2 MHz where the white rectangle in front of the bregma towards the nose shows the acoustic window through which NAc is accessible. D (D1) 10 element 1D array. Azimuthal cross section of the array and the two stimulation scenarios: (D2) simultaneous stimulation, (D3) sequential stimulation: beam pattern at 2.4 MHz behind an ex vivo rat skull while simultaneously targeting the two NAc locations
Experiment flowchart. EG: experiment (stimulation) group; STS: short-term stimulation group; LTS: long-term stimulation group; SG: sham group
mRNA microarray expression profile results. A Expression change set according to the sham group in STS (short-term stimulation group), in the NAc (nucleus accumbens): 454 genes with changed expression are shown. The color scale indicates the expression rate. B Expression change set according to the sham group in LTS (long-term stimulation group), in the NAc: 382 genes with changed expression are shown. The color scale indicates the expression rate. C Expression change cluster in LTS toward STS: 32 genes that vary in expression are shown. The color scale indicates the expression rate
MicroRNAs with reduced regulation and validation. Relation between miRNA-mRNA after validation and pathways involved in these genes (orange texts represent STS, gray texts represent LTS pathways) (K: sham; A: STS (short-term stimulation group); B: LTS (long-term stimulation group)
Histological findings. A Anti-map2 immunohistochemical staining of the groups. STS (short-term stimulation group) (A1), LTS (long-term stimulation group) (A2), and sham (A3), respectively, in the nucleus accumbens region (NAc) of brain tissue sections. Microtubules stained with anti-map2 are seen in red color. The nuclei were stained with DAPI (blue). There was no difference in intensity of anti-map2 staining between the groups in the comparison of red fluorescence radiation intensity. B Anti-JNK1 immunohistochemical staining in NAc in the brain tissue sections of STS (B1), LTS (B2), and sham (B3). As a result of staining, there was no JNK1-positive cell in the NAc region in any group. Blood vessel was stained for positive control of anti-JNK1 antibody staining (B4). Nuclei stained with DAPI (blue) and anti-JNK1-positive cells in blood vessel wall (green) are seen. C TUNEL method staining in brain tissue sections in NAc of STS (C1), LTS (C2), and sham (C3) groups. No damaged cells (TUNEL + cell) were detected in the NAc region. For positive control of TUNEL method staining, brain tissue sections were treated with DNase and DNA breaks were generated (C4). Cells with DNA damage are looking green (TUNEL +). Normal cells were stained with DAPI (TUNEL −), and those cells are looking blue (× 40/1, 4 oil immersion objective, scale: 20 μm)
We investigated the effect of low-intensity focused ultrasound (LIFU) on gene expression related to alcohol dependence and histological effects on brain tissue. We also aimed at determining the miRNA-mRNA relationship and their pathways in alcohol dependence-induced expression changes after focused ultrasound therapy. We designed a case–control study for 100 days of observation to investigate differences in gene expression in the short-term stimulation group (STS) and long-term stimulation group (LTS) compared with the control sham group (SG). The study was performed in our Experimental Research Laboratory. 24 male high alcohol-preferring rats 63 to 79 days old, weighing 270 to 300 g, were included in the experiment. LTS received 50-day LIFU and STS received 10-day LIFU and 40-day sham stimulation, while the SG received 50-day sham stimulation. In miRNA expression analysis, it was found that LIFU caused gene expression differences in NAc. Significant differences were found between the groups for gene expression. Compared to the SG, the expression of 454 genes in the NAc region was changed in the STS while the expression of 382 genes was changed in the LTS. In the LTS, the expression of 32 genes was changed in total compared to STS. Our data suggest that LIFU targeted on NAc may assist in the treatment of alcohol dependence, especially in the long term possibly through altering gene expression. Our immunohistochemical studies verified that LIFU does not cause any tissue damage. These findings may lead to new studies in investigating the efficacy of LIFU for the treatment of alcohol dependence and also for other psychiatric disorders.
 
Sleep loss is often associated with cognitive dysfunction. Alterations in the structure and function of synapses in the hippocampus are thought to underlie memory storage. Paired immunoglobulin-like receptor B (PirB) plays a negative role in various neurological diseases by inhibiting axon regeneration and synaptic plasticity. However, the contributions of PirB to the mechanisms underlying the changes in synaptic plasticity after sleep loss that ultimately promote deficits in cognitive function have not been well elucidated. Here, we showed that chronic sleep restriction (CSR) mice displayed cognitive impairment and synaptic deficits accompanied by upregulation of PirB expression in the hippocampus. Mechanistically, PirB caused the dysregulation of actin through the RhoA/ROCK2/LIMK1/cofilin signalling pathway, leading to abnormal structural and functional plasticity, which in turn resulted in cognitive dysfunction. PirB knockdown alleviated synaptic deficits and cognitive impairment after CSR by inhibiting the RhoA/ROCK2/LIMK1/cofilin signalling pathway. Moreover, we found that fasudil, a widely used ROCK2 inhibitor, could mimic the beneficial effect of PirB knockdown and ameliorate synaptic deficits and cognitive impairment, further demonstrating that PirB induced cognitive dysfunction after CSR via the RhoA/ROCK2/LIMK1/cofilin signalling pathway. Our study sheds new light on the role of PirB as an important mediator in modulating the dysfunction of synaptic plasticity and cognitive function via the RhoA/ROCK2/LIMK1/cofilin signalling pathway, which indicated that hippocampal PirB is a promising therapeutic target for counteracting cognitive impairment after CSR. Graphical Abstract This illustration depicts the signalling pathway by PirB in mediating cognitive impairment and synaptic deficits in CSR mice. In the hippocampus of CSR mice, the expression level of PirB was significantly increased. In addition, CSR increases RhoA and ROCK2 levels and reduces levels of both LIMK1 and cofilin phosphorylation. PirB knockdown reverses cognitive impairment and synaptic plasticity disorders caused by CSR through the RhoA/ROCK2/LIMK1/cofilin signalling pathway
 
Intracerebral hemorrhage (ICH) is characterized by poor prognosis and high mortality rates. To date, satisfactory therapeutic approaches for ICH remain limited, so it is urgently needed to develop a safer and more effective prescription. Secondary inflammatory response has been acknowledged as an aggravating factor to neurological deterioration after ICH. As a component of inflammasome sensors, absent in melanoma 2 (AIM2) plays an important role in the neuroinflammation process. Here, ozanimod, a novel selective sphingosine 1-phosphate receptor modulator, has gained much attention, which alleviates the resultant neuroinflammation and improves functional recovery derived from ICH. In this study, ozanimod improved neurological functions of ICH mice via reduction of hematoma size. Furthermore, both microglial and AIM2 inflammasome activations were reversed by ozanimod, which are confirmed by the downregulation of related inflammatory proteins and cytokines (IL-1β, IL-6, and TNF-α), coupled with the upregulation of SIRT3, by leveraging the Western blot and enzyme-linked immunosorbent assay. Additionally, we find that ozanimod decreases nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression. Notably, in vitro cell experiments induced by lipopolysaccharide confirms that the anti-inflammatory effect of ozanimod could be abolished by the SIRT3 inhibitor. In conclusion, these results indicate that ozanimod mitigates ICH-induced secondary inflammatory responses by modulating AIM2 inflammasome mediated by SIRT3/NF-κB/AIM2 pathway. This demonstrates ozanimod orchestrates ICH-induced neuroinflammation and could be a targeted therapy for improving prognosis of ICH.
 
Confirmation of next-generation sequencing results using qRT-PCR. Data expressed as mean RQ with standard deviation (n = 8 per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus sham control (Student’s t-test). Filled diamond = Sham ECS group; Unfilled diamond = ECS treated group
Correlations between whole blood miRNAs and mood scores. a Correlation between baseline whole blood miR-204 and baseline HAM-D24. b Correlation between baseline whole blood miR-204 and the change in HAM-D24 post-ECT
Correlations between CSF and serum miRNAs and mood score and duration of current episode. a Correlation between baseline CSF miR-204 and the duration of the current depressive episode in weeks. b Correlation between the change in serum miR-212 and the change in HAM-D21
MicroRNAs (miRNAs) may contribute to the development of depression and its treatment. Here, we used the hypothesis-neutral approach of next-generation sequencing (NGS) to gain comprehensive understanding of the effects of a course of electroconvulsive stimulation (ECS), the animal model equivalent of electroconvulsive therapy (ECT), on rat hippocampal miRNAs. Significant differential expression (p < 0.001) of six hippocampal miRNAs was noted following NGS, after correcting for multiple comparisons. Three of these miRNAs were upregulated (miR-132, miR-212, miR-331) and three downregulated (miR-204, miR-483, miR-301a). qRT-PCR confirmed significant changes in four of the six miRNAs (miR-132, miR-212, miR-204, miR-483). miR-483 was also significantly reduced in frontal cortex, though no other significant alterations were noted in frontal cortex, cerebellum, or whole blood. Assessing the translatability of the results, miR-132 and miR-483 were significantly reduced in whole blood samples from medicated patients with depression (n = 50) compared to healthy controls (n = 45), though ECT had no impact on miRNA levels. Notably, pre-ECT miR-204 levels moderately positively correlated with depression severity at baseline and moderately negatively correlated with mood score reduction post-ECT. miRNAs were also examined in cerebrospinal fluid and serum from a separate cohort of patients (n = 8) treated with ECT; no significant changes were noted post-treatment. However, there was a large positive correlation between changes in miR-212 and mood score post-ECT in serum. Though replication studies using larger sample sizes are required, alterations in miRNA expression may be informative about the mechanism of action of ECS/ECT and in turn might give insight into the neurobiology of depression.
 
Transcriptome of the dorsolateral prefrontal cortex in humans with schizophrenia. A Principal component analysis score plots displaying the discrimination of the transcriptome in the dorsolateral prefrontal cortex (DLPFC) of males (n = 160) and females (n = 93) with schizophrenia and male (n = 154) and female (n = 113) controls. SCZ, schizophrenia. B Rank–rank hypergeometric overlap (RRHO) shows overlapping differentially upregulated and downregulated genes affected by schizophrenia between males and females. C Venn diagram summarizing the number of genes altered in the DLPFC of males and females with schizophrenia compared with male and female controls. D The top 10 most enriched biological process categories in female schizophrenia-related genes based on DAVID analyses. GO, Gene Ontology
Maternal immune activation-induced transcriptional changes in the offspring. A Prepulse test of offspring following maternal immune activation (MIA) exposure (male, n = 10; female, n = 10). B Prepulse test of offspring following MIA exposure in female (n = 10). C Prepulse test of offspring following MIA exposure in female (n = 10). D Drawing of a section of the prefrontal cortex (PFC, red area) and sectioned region of brain tissue. M2, secondary motor cortex; OB, olfactory bulb. (E) Venn diagram summarizing the number of genes altered in the PFC of male offspring (n = 3) and female offspring (n = 160) following MIA compared with male (n = 3) and female (n = 3) controls. F Rank–rank hypergeometric overlap (RRHO) shows overlapping of differentially upregulated and downregulated genes in response to MIA exposure between males and female offspring
Gene expression and methylation of ACSBG1 in females. A, B Expression levels of Acsbg1 in the mouse PFC of female (A) and male (B) offspring were measured by real-time qRT-PCR (n = 10). C Expression levels of ACSBG1 in the human PFC of females with schizophrenia (SCZ, n = 11) and female controls (health, n = 10) were measured by real-time qRT-PCR. SCZ, schizophrenia. D Amino acid sequence of mouse ACSBG1 (exon 8) compared with human ACSBG1 (exon 8). Different amino acids are highlighted in red. E Analysis of Acsbg1 DNA methylation levels in female mouse offspring by bisulfite pyrosequencing. Upper: fragment (178 bp) in hypermethylated peak 4 in female offspring (Supplementary Fig. 7). Exon 8 is highlighted in red. CpG sites are highlighted in yellow. Lower: average percent of methylation levels in each CpG site (n = 10). F Analysis of ACSBG1 DNA methylation levels in female patients with schizophrenia (n = 9) and female controls (n = 9) by bisulfite pyrosequencing. Upper: bisulfite sequencing-amplified 178-bp fragment in human ACSBG1 exon 8. CpG sites are highlighted in yellow. Lower: average percent of methylation levels in each CpG site. All data are expressed as the mean ± SEM. *P < 0.05
Schizophrenia presents clinical and biological differences between males and females. This study investigated transcriptional profiles in the dorsolateral prefrontal cortex (DLPFC) using postmortem data from the largest RNA-sequencing (RNA-seq) database on schizophrenic cases and controls. Data for 154 male and 113 female controls and 160 male and 93 female schizophrenic cases were obtained from the CommonMind Consortium. In the RNA-seq database, the principal component analysis showed that sex effects were small in schizophrenia. After we analyzed the impact of sex-specific differences on gene expression, the female group showed more significantly changed genes compared with the male group. Based on the gene ontology analysis, the female sex-specific genes that changed were overrepresented in the mitochondrion, ATP (phosphocreatine and adenosine triphosphate)-, and metal ion-binding relevant biological processes. An ingenuity pathway analysis revealed that the differentially expressed genes related to schizophrenia in the female group were involved in midbrain dopaminergic and γ-aminobutyric acid (GABA)-ergic neurons and microglia. We used methylated DNA-binding domain-sequencing analyses and microarray to investigate the DNA methylation that potentially impacts the sex differences in gene transcription using a maternal immune activation (MIA) murine model. Among the sex-specific positional genes related to schizophrenia in the PFC of female offspring from MIA, the changes in the methylation and transcriptional expression of loci ACSBG1 were validated in the females with schizophrenia in independent postmortem samples by real-time PCR and pyrosequencing. Our results reveal potential genetic risks in the DLPFC for the sex-dependent prevalence and symptomology of schizophrenia.
 
Curcumin (CUR) and piperine (PIP) are very well-known phytochemicals that claimed to have many health benefits and have been widely used in foods and traditional medicines. This study investigated the therapeutic efficacy of these compounds to treat Alzheimer’s disease (AD). However, poor oral bioavailability and permeability of curcumin are a major challenge for formulation scientists. In this research study, the researcher tried to enhance the bioavailability and permeability of curcumin by a nanotechnological approach. In this research study, we developed a CUR–PIP-loaded SNEDDS in various oils. Optimised formulation NF3 was subjected to evaluate its therapeutic effectiveness on AD animal model in comparison with untreated AD model and treated group (by market formulation donepezil). On the basis of characterisation results, it is confirmed that NF3 formulation is the best formulation. The optimised formulation shows a significant dose-dependent manner therapeutic effect on AD-induced model. Novel formulation CUR–PIP solid-SNEDDS was successfully developed and optimised. It is expected that the developed S-SNEDDS can be a potential, safe and effective carrier for the oral delivery of curcumin to the brain. To date, this article is the only study of CUR–PIP-loaded S-SNEDDS for the treatment of AD.
 
PtdSer regulates AKT and PKC signaling. Schematic demonstrating that PtdSer regulates AKT and PKC signaling by maintaining their membrane location. The PH domain present in the AKT protein possesses basic residues R15/K20 that bind directly to PtdSer, facilitating contact between PIP3 and AKT. The regulatory domain in AKT can also bind directly to PtdSer via the basic residues K419/K420. PKC has two domains that can bind to cytoplasmic PtdSer: the C1 domain binds to PtdSer in a stereotaxic manner, while the C2 domain binds to PtdSer in a Ca²⁺-independent stereo-specific manner. Activated AKT and PKC further promote cell survival and cell growth via regulating downstream signaling molecules [34, 35]. PH domain: Pleckstrin Homology domain; GSK3β: glycogen synthase kinase-3 beta; TSC: tuberous sclerosis complex; RAF: Raf kinase; mTOR: mammalian target of rapamycin; MEK: mitogen-activated protein kinase; ERK: extracellular signal-regulated kinase
The P4-ATPase–CDC50A complex flips PtdSer. The proper function of P4-ATPases is dependent on heterodimerization with CDC50A. P4-ATPases have three cytosolic domains: the actuator (A) domain, the nucleotide-binding (N) domain, and the phosphorylation (P) domain. P4-ATPases use energy from ATP hydrolysis within their N domain to mediate PtdSer translocation. P-type ATPase-coupled transport involves the formation and hydrolysis of several phosphoenzyme intermediate states (E1, E1P, E2P, and E2), which is described by the Post-Albers cycle. The P4-ATPase transport cycle proceeds as follows: (1) binding of ATP slightly shifts the N-domain of P4-ATPases from E1 state to the E1-ATP state; (2) phosphorylation of the P-domain shifts the N-domain upward to yield the E1P-ADP state. This process in turn shifts the A-domain slightly toward the N-terminus of Cdc50a, stabilizing the conformation; (3) through ADP release, the A-domain shifts toward the phosphorylation site, which opens the lipid transport pathway and results in the E2P state; (4) binding of PtdSer results in closure of the lipid entry site; and (5) release of PtdSer and Pi returns the P4-ATPase-CDC50A complex to the E1 state. This model schematic is constructed according to reference [58, 59]
Microglial phagocytosis of synapses, neurons, and pathological proteins through exposed PtdSer. PtdSer exposure occurs in certain neurodegenerative diseases, since neurofibrillary tangles accelerate cytoplasmic ROS generation, causing the occurrence of PtdSer exposure at the outer leaflet of the plasma membrane in live neurons. Subsequently, microglia phagocytose live neurons, which exacerbates neurodegeneration. Moreover, extracellular Aβ plaques are decorated with exposed PtdSer, which can be engulfed by microglia through TAM receptors. During brain development, microglia transiently contact PtdSer-exposed synapses, which initiates synaptic pruning to eliminate supernumerary synapses.
Microglia recognize exposed PtdSer via different receptors. Microglia express different PtdSer receptors that bind to exposed PtdSer either directly or indirectly. The figure illustrates different PtdSer receptors on microglia and their potential downstream signal transduction, which regulates microglial polarization and cytoskeletal rearrangement contributing to phagocytosis. BAI1, brain-specific angiogenesis inhibitor-1; C1q, complement C1q; CD36, cluster of differentiation 36; CR3, complement receptor type 3; DAP12, DNAX activation protein 12; DOCK180, downstream of Crk family member 180I; ELMO, engulfment and cell motility proteins; Gas6, growth-arrest-specific 6; GPR56, adhesion G protein-coupled receptor G1; JAK, the Janus kinase; MFGE8, milk fat globule epidermal growth factor 8; Rac1, Ras-related C3 botulinum toxin substrate 1; RAGE, receptor for advanced glycation end-products; STAT, signal transducer of activation proteins; SOCS1/3, suppressor of cytokine signaling 1/3; TAM receptors, receptor tyrosine kinases TYRO3, AXL, and MER; TIM receptors, T cell/transmembrane, immunoglobulin, and mucin family receptors; TREM2, triggering receptor expressed on myeloid cells 2
Phosphatidylserine (PtdSer) is an important anionic phospholipid found in eukaryotic cells and has been proven to serve as a beneficial factor in the treatment of neurodegenerative diseases. PtdSer resides in the inner leaflet of the plasma membrane, where it is involved in regulating the AKT and PKC signaling pathways; however, it becomes exposed to the extracellular leaflet during neurodevelopmental processes and neurodegenerative diseases, participating in microglia-mediated synaptic and neuronal phagocytosis. In this paper, we review several characteristics of PtdSer, including the synthesis and translocation of PtdSer, the functions of cytoplasmic and exposed PtdSer, and different PtdSer-detection materials used to further understand the role of PtdSer in the nervous system.
 
Intermittent access of 20% ethanol drinking paradigm in SD rats is constructed. A A Schematic picture of experimental designs. IA2BC induced stable ethanol consumption in male rats (B) and preference (C). D There was no difference in body weight between alcohol and control groups (F1,10 = 1.307, P = 0.2795, two-way ANOVA). E There was no difference in total fluid consumption between alcohol and control groups (t = 0.6624, P = 0.5263, unpaired Student’s t-test). Data are expressed as mean ± standard error of the mean (SEM). The number of rats per group = 10 (B, C), number of rats per group = 6 (D, E)
Ethanol decreased KCC2 expression after 4 weeks of ethanol consumption. A The amount of ethanol intake 72 h after withdrawal exceeded the last ethanol consumption and ethanol intake after other withdrawal times in IA2BC rats (F4,25 = 3.975, P = 0.0125, one-way ANOVA and Tukey’s post hoc test). Western blot analysis was conducted for total KCC2 and phosphorylated-Ser940 KCC2 with GAPDH as a loading control. B It represents that western blot indicates reduced expression of pS940 KCC2 relative to total KCC2 in IA2BC rats when compared to controls. Gray value analysis revealed a significant reduction in pKCC2(C) and tKCC2 (D), *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group. Gray value analysis revealed a reduction in the ratio of Ser940 KCC2 to total KCC2 protein 72 h after withdrawal, *P < 0.05 (E). Data are expressed as mean ± standard error of the mean (SEM). Number of rats per group = 6 (A). Number of rats per group = 4 (C, D, E)
KCC2 decreased voluntary ethanol consumption in IA2BC rats after alcohol withdrawal. A Compared with the vehicle group, both CLP290 (50 μM) and CLP290 (100 μM) significantly reduced ethanol intake (F4,25 = 7.761, P(vehicle vs.CLP290(50 μM)) = 0.0023, and P(vehicle vs.CLP290(100 μM)) = 0.0161, one-way ANOVA, and Tukey’s post hoc test). B Both microinjections of VU0240551 (10 μM) and VU0240551 (30 μM) into the VTA increased ethanol intake (F4,25 = 5.701, P(vehicle vs.vu0240551(10 μM)) = 0.0258, P(vehicle vs.vu0240551(30 μM)) = 0.0025,one-way ANOVA, and Tukey’s post hoc test). C It represents a picture of bilateral VTA microinfusion of drugs that was briefly described. Data are expressed as mean ± standard error of the mean (SEM). Number of rats per group = 6. *P < 0.05, **P < 0.01, compared to the vehicle
KCC2 may be involved in the regulation of BDNF-TrkB on alcohol consumption in IA2BC rats. A Microinjection of VU0240551 (10 μM) into VTA blocked the effects of BDNF on ethanol intake (F2,15 = 12.30, P = 0.0045, one-way ANOVA, and Tukey’s post hoc test), **P < 0.01, compared to the vehicle. B Microinjection of CLP290 (50 μM) into VTA reduced ethanol intake again after blocking effect of 7,8-DHF using K252a at 72 h after withdrawal (F3,20 = 8.064, P < 0.001, one-way ANOVA, and Tukey’s post hoc test), **P < 0.01compared to the sham, ****P < 0.0001 compared to the control. C Western blot analysis was conducted for total KCC2 and phosphorylated-Ser940 KCC2 protein expression, with GAPDH as a loading control. 7,8-DHF (5 mg/kg) significantly increased the expression of pKCC2 (D) and slightly increased expression of tKCC2 at 72-h withdrawal in IA2BC rats (E). *P < 0.05, **P < 0.01 compared to the control group. Data are expressed as mean ± standard error of the mean (SEM). Number of rats per group = 6
Alcohol use disorder (AUD) is a common and complex disorder resulting from repetitive alcohol drinking. The mesocorticolimbic dopamine (DA) system, originating from the ventral tegmental area (VTA) in the midbrain, is involved in the rewarding effect of ethanol. The γ-aminobutyric acid (GABA) neurons in VTA appear to be key substrates of acute and chronic ethanol, which regulates DA neurotransmission indirectly in the mesocorticolimbic system. Despite significant research on the relationship between brain-derived neurotrophic factor (BDNF) and reduced alcohol consumption in male rats involving tropomyosin-related kinase B (TrkB), the mechanisms of BDNF-TrkB regulating alcohol behavior remain scarce. K+-Cl– cotransporter 2 (KCC2) plays a crucial role in synaptic function in GABAergic neurons by modulating intracellular chlorine homeostasis. Here, we found that 4-week intermittent alcohol exposure impaired the function of KCC2 in VTA, evidenced by a lower expression level of phosphorylated KCC2 and decreased ratio of phosphorylated KCC2 to total KCC2, especially 72 h after withdrawal from 4-week ethanol exposure in male rats. CLP290 (a KCC2 activator) reduced excessive alcohol consumption after alcohol withdrawal, whereas VU0240551 (a specific KCC2 inhibitor) further enhanced alcohol intake. Importantly, VU0240551 reversed the attenuating effects of BDNF and 7,8-dihydroxyflavone (7,8-DHF) on alcohol consumption after withdrawal. Moreover, intraperitoneal injection of 7,8-DHF upregulated KCC2 expression and phosphorylated KCC2 in VTA 72 h after withdrawal from ethanol exposure in male rats. Collectively, our data indicate that KCC2 may be critical in the regulating action of BDNF-TrkB on ethanol consumption in AUD.
 
In Alzheimer disease (AD), Tau, an axonal microtubule-associated protein, becomes hyperphosphorylated, detaches from microtubules, accumulates, and self-aggregates in the somatodendritic (SD) compartment. The accumulation of hyperphosphorylated and aggregated Tau is also seen in other neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD-Tau). Previous studies reported a link between filamin A (FLNA), an actin-binding protein found in the SD compartment, and Tau pathology. In the present study, we further explored this link. We confirmed the interaction of Tau with FLNA in neuroblastoma 2a (N2a) cells. This interaction was mediated by a domain located between the 157 and 383 amino acids (a.a.) of Tau. Our results also revealed that the overexpression of FLNA resulted in an intracellular accumulation of wild-type Tau and Tau mutants (P301L, V337M, and R406W) in N2a cells. Tau phosphorylation and cleavage by caspase-3 but not its aggregation were increased upon FLNA overexpression in N2a cells. In the parietal cortex of AD brain, insoluble FLNA was increased compared to control brain, but it did not correlate with Tau pathology. Interestingly, Tau binding to microtubules and F-actin was preserved upon FLNA overexpression in N2a cells. Lastly, our results revealed that FLNA also induced the accumulation of annexin A2, a Tau interacting partner involved in its axonal localization. Collectively, our data indicated that in Tauopathies, FLNA could contribute to Tau pathology by acting on Tau and annexin A2.
 
Parkinson’s disease (PD) is characterized by progressive loss of dopaminergic neurons and accumulation of misfolded alpha-synuclein (αSyn) into Lewy bodies. In addition to motor impairment, PD commonly presents with cognitive impairment, a non-motor symptom with poor outcome. Cortical αSyn pathology correlates closely with vascular risk factors and vascular degeneration in cognitive impairment. However, how the brain microvasculature regulates αSyn pathology and neurodegeneration remains unclear. Here, we constructed a rapidly progressive PD model by injecting alpha-synuclein preformed fibrils (αSyn PFFs) into the cerebral cortex and striatum. Brain capillaries in mice with cognitive impairment showed a reduction in diameter and length after 6 months, along with string vessel formation. The intracellular domain of low-density lipoprotein receptor-related protein-1 (LRP1-ICD) was upregulated in brain microvascular endothelium. LRP1-ICD promoted αSyn PFF uptake and exacerbated endothelial damage and neuronal apoptosis. Then, we overexpressed LRP1-ICD in brain capillaries using an adeno-associated virus carrying an endothelial-specific promoter. Endothelial LRP1-ICD worsened αSyn PFF-induced vascular damage, αSyn pathology, or neuron death in the cortex and hippocampus, resulting in severe motor and cognitive impairment. LRP1-ICD increased the synthesis of poly(adenosine 5′-diphosphate-ribose) (PAR) in the presence of αSyn PFFs. Inhibition of PAR polymerase 1 (PARP1) prevented vascular-derived injury, as did loss of PARP1 in the endothelium, which was further implicated in endothelial cell proliferation and inflammation. Together, we demonstrate a novel vascular mechanism of cognitive impairment in PD. These findings support a role for endothelial LRP1-ICD/PARP1 in αSyn pathology and neurodegeneration, and provide evidence for vascular protection strategies in PD therapy.
 
The detrimental impact of fructose, a widely used sweetener in industrial foods, was previously evidenced on various brain regions. Although adolescents are among the highest consumers of sweet foods, whether brain alterations induced by the sugar intake during this age persist until young adulthood or are rescued returning to a healthy diet remains largely unexplored. To shed light on this issue, just weaned rats were fed with a fructose-rich or control diet for 3 weeks. At the end of the treatment, fructose-fed rats underwent a control diet for a further 3 weeks until young adulthood phase and compared with animals that received from the beginning the healthy control diet. We focused on the consequences induced by the sugar on the main neurotrophins and neurotransmitters in the frontal cortex, as its maturation continues until late adolescence, thus being the last brain region to achieve a full maturity. We observed that fructose intake induces inflammation and oxidative stress, alteration of mitochondrial function, and changes of brain-derived neurotrophic factor (BDNF) and neurotrophin receptors, synaptic proteins, acetylcholine, dopamine, and glutamate levels, as well as increased formation of the glycation end-products Nε -carboxymethyllysine (CML) and Nε -carboxyethyllysine (CEL). Importantly, many of these alterations (BDNF, CML, CEL, acetylcholinesterase activity, dysregulation of neurotransmitters levels) persisted after switching to the control diet, thus pointing out to the adolescence as a critical phase, in which extreme attention should be devoted to limit an excessive consumption of sweet foods that can affect brain physiology also in the long term.
 
Treadmill exercise is widely considered an effective strategy for restoration of skilled motor function after spinal cord injury (SCI). However, the specific exercise intensity that optimizes recovery and the underlying mechanistic basis of this recovery remain unclear. To that end, we sought to investigate the effect of different treadmill exercise intensities on cortical mTOR activity, a key regulator of functional recovery following CNS trauma, in an animal model of C5 crush spinal cord injury (SCI). Following injury, animals were subjected to treadmill exercise for 4 consecutive weeks at three different intensities (low intensity [LEI]; moderate intensity [MEI]; and high intensity [HEI]). Motor function recovery was assessed by horizontal ladder test, cylinder rearing test, and electrophysiology, while neurotrophic factors and cortical mechanistic target of rapamycin (mTOR) pathway–related proteins were assessed by Western blotting. The activation of the cortical mTOR pathway and axonal sprouting was evaluated by immunofluorescence and the changes of plasticity in motor cortex neurons were assessed by Golgi staining. In keeping with previous studies, we found that 4 weeks of treadmill training resulted in improved skilled motor function, enhanced nerve conduction capability, increased neuroplasticity, and axonal sprouting. Importantly, we also demonstrated that when compared with the LEI group, MEI and HEI groups demonstrated elevated expression of brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), phosphorylated ribosomal S6 protein (p-S6), and protein kinase B (p-Akt), consistent with an intensity-dependent activation of the mTOR pathway and neurotrophic factor expression in the motor cortex. We also observed impaired exercise endurance and higher mortality during training in the HEI group than in the LEI and MEI groups. Collectively, our findings suggest that treadmill exercise following SCI is an effective means of promoting recovery and highlight the importance of the cortical mTOR pathway and neurotrophic factors as mediators of this effect. Importantly, our findings also demonstrate that excessive exercise can be detrimental, suggesting that moderation may be the optimal strategy. These findings provide an important foundation for further investigation of treadmill training as a modality for recovery following spinal cord injury and of the underlying mechanisms.
 
Microcystin-LR (MC-LR) has been confirmed to cause blood–brain barrier disruption and enter the brain tissue, resulting in non-negligible toxic effects. However, the neurotoxicity of MC-LR is mainly unknown. This study revealed that MC-LR disrupted the function of the ubiquitin–proteasome system in neurons, which inhibited the degradation of α-synuclein (α-syn), leading to its release from neurons for transport into microglia. α-Syn is the main component of Lewy bodies, which has been identified as one of the main pathological features of Parkinson’s disease (PD). In vitro, we observed that α-syn mediated by MC-LR activated HMC3 cells and polarized them towards M1 type. In addition, we confirmed that α-syn was transported into HMC3 cells through TLR4 receptors and activated the NLRP3 inflammasome, which in turn enhanced the maturation and release of IL-18 and IL-1β. In the mouse models of chronic MC-LR exposure, a large number of inflammatory factors (IL-6, IL-1β, and TNF-α) were deposited in brain tissue, and activation of NLRP3 in microglia was also observed in the midbrain. Collectively, MC-LR exposure promoted the pathological spread of α-syn from cell to cell, activated NLRP3 inflammasome in microglia, and generated neuroinflammation, in which the TLR4 receptor played a substantial effect.
 
3D sample image graphics of MS patient and healthy control samples
Normalization image of MS patient and healthy control groups
Bioinformatics analysis of MS patients and healthy controls
In our study, we aimed to investigate the relationship between microRNA (miRNA) expression levels and serum iron (Fe), copper (Cu), and zinc (Zn) levels in Multiple sclerosis (MS) patients. Total RNA was isolated from peripheral venous blood containing ethylenediaminetetraacetic acid (EDTA) of MS patients and controls. Total RNA was labeled with Cy3-CTP fluorescent dye. Hybridization of samples was performed on microarray slides and arrays were scanned. Data argument and bioinformatics analysis were performed. Atomic absorption spectrophotometer method was used to measure serum Fe, Cu, and Zn levels. In our study, in bioinformatics analysis, although differently expressed miRNAs were not detected between 16 MS patients and 16 controls, hsa-miR-744-5p upregulation was detected between 4 MS patients and 4 controls. This may be stem from the patient group consisting of MS patients who have never had an attack for 1 year. Serum iron levels were detected significantly higher in the 16 MS patients compared to the 16 controls. This may be stem from the increase in iron accumulation based on inflammation in MS disease. According to the findings in our study, hsa-miR-744-5p upregulation has been determined as an early diagnostic biomarker for the development together of insulin resistance, diabetes mellitus associated with insulin signaling, and Alzheimer’s diseases. Therefore, hsa-miR-744-5p is recommended as an important biomarker for the development together of diabetes mellitus, Alzheimer’s disease, and MS disease. In addition, increased serum Fe levels may be suggested as an important biomarker for neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and MS disease.
 
Neurons deficient in the TLR4 receptor are resistant to OGD-induced apoptosis and LPS-induced intracellular Ca²⁺ increase. a, b Cultured cortical neurons from WT mice and TLR4 knockout mice were exposed to oxygen and glucose deprivation for 30 min, and lactate dehydrogenase (LDH) release (a) and cell viability (b) were measured (n = 5). c, d Cell survival was quantified by TUNEL staining following OGD treatment. e Cultured neurons were exposed to fluo-4 AM, and the changes in the intracellular Ca²⁺ levels were evaluated using confocal microscopy. The green fluorescence (fluo-4 AM) intensity, which represents the [Ca²⁺]i (intracellular calcium) concentration, changes time-dependently in neurons. f [Ca²⁺]i was measured for 300 s in five independent experiments. The results showed that the average kinetics of Ca²⁺ in the WT + LPS groups were greater than those in the TLR4−/− + LPS groups (n = 5). LPS treatment is shown by arrows. g Bar graphs represent the peak value of the neuronal Ca.²⁺ response to LPS treatment (n = 5). Values are represented as the mean ± SEM. **P < 0.01
Deletion of TLR4 from glutamatergic neurons protects neurons from ischemic damage and improves functional deficits in a mouse stroke model. a–d Images of the cortex, striatum, and hippocampus from TLR4cKO (Vglut2ires−Cre/+ + Tlr4fl/fl) and control (Tlr4fl/fl) mice that were stained with TUNEL (a) and Fluoro-Jade B (FJB) (c) 24 h after ischemia/reperfusion (I/R). Bar graphs show summarized TUNEL-labeled (b) and FJB-labeled (d) cells (n = 5). e Images of brain sections from TLR4.cKO and control mice 24.5 h after I/R were stained with TTC. f, g Bar graphs show infarct volumes (f) (n = 5) and neurological scores (g) (n = 10) for mice subjected to I/R. Values are represented as the mean ± SEM. *P < 0.05, **P < 0.01
Colocalization of TLR4 and NMDAR2B in neurons respond to ischemic stimulation. a, b Representative immunofluorescence photomicrographs showing the colocalization of NMDAR2B (purple) and TLR4 (red) in neurons (green) in vitro (a) and in vivo (b) following OGD and I/R, respectively
Involvement of Src kinase and the NMDAR2B phosphorylation axis in TLR4-mediated neuronal death following OGD and LPS-induced intracellular Ca²⁺ increase. a Immunoblot analysis of proteins in cell lysates of neurons from WT and TLR4 knockout mice following exposure to OGD. b–d Bar graphs show the levels of phosphorylation at Ser-1303 (p-NMDAR2B)/NMDAR2B (b), nNOS (c), and Src kinase (d) (n = 4). e, f PP2, an inhibitor of the Src family of protein kinases, effectively decreased lactate dehydrogenase (LDH) release (e) and increased cell viability (f) following OGD (n = 5). g, h Cell survival was quantified using TUNEL staining following PP2 and OGD treatments (n = 5). i Blots showing NMDAR2B, p-NMDAR2B, and nNOS in cell lysates of neurons following PP2 and OGD treatments. j, k Bar graphs show the levels of phosphorylation at Ser-1303 (p-NMDAR2B)/NMDAR2B (j) and nNOS (k) (n = 4). l The average kinetics of Ca²⁺ in the WT + LPS and WT + LPS + PP2 groups (n = 5). LPS treatment is shown by arrows. m Bar graphs represent the peak value of the neuronal Ca.²⁺ response for LPS and PP2 treatments (n = 5). Values are represented as the mean ± SEM. *P < 0.05, **P < 0.01
Deletion of TLR4 from glutamatergic neurons decreases the phosphorylation of NMDAR2B and dissociates the nNOS–PSD-95 interaction in a mouse stroke model. a Western blot images of protein bands. b–d Quantitative analysis of the levels of phosphorylation at Ser-1303 (p-NMDAR2B)/NMDAR2B (b), nNOS (c), and Src kinase (d) (n = 4). e, f Coimmunoprecipitation experiments showing the effect of deletion of TLR4 from glutamatergic neurons on nNOS–PSD-95 interaction after I/R (n = 5). Values represent the mean ± SEM. **P < 0.01
In microglia, Toll-like receptor 4 (TLR4) is well known to contribute to neuroinflammatory responses following brain ischemia. TLR4 is also expressed in neurons and can mediate the conduction of calcium (Ca²⁺) influx, but the mechanistic link between neuronal TLR4 signaling and brain ischemic injury is still poorly understood. Here, primary neuronal cell cultures from TLR4 knockout mice and mice with conditional TLR4 knockout in glutamatergic neurons (TLR4cKO) were used to establish ischemic models in vitro and in vivo, respectively. We found that deleting TLR4 would reduce the neuronal death and intracellular Ca²⁺ increasement induced by oxygen and glucose deprivation (OGD) or lipopolysaccharide treatment. Infarct volume and functional deficits were also alleviated in TLR4cKO mice following cerebral ischemia/reperfusion (I/R). Furthermore, TLR4 and N-methyl-d-aspartate receptor subunit 2B (NMDAR2B) were colocalized in neurons. Deletion of TLR4 in neurons rescued the upregulation of phosphorylated NMDAR2B induced by ischemia via Src kinase in vitro and in vivo. Downstream of NMDAR2B signaling, the interaction of neuronal nitric oxide synthase (nNOS) with postsynaptic density protein-95 (PSD-95) was also disrupted in TLR4cKO mice following cerebral I/R. Taken together, our results demonstrate a novel molecular neuronal pathway in which TLR4 signaling in neurons plays a crucial role in neuronal death and provide a new target for neuroprotection after ischemic stroke.
 
The effects of M1 and M2 microglia on neuroinflammation and neurogenesis. M1 microglia plays significant roles in secreting pro-inflammatory cytokines like IL6, IL1B, IL12, CCL2, TNF-a, and ROS. They also exert anti-neurogenic effects by decreasing NSC proliferation and NeuN + cells. On the other side, M2 microglia have active roles in secreting anti-inflammatory cytokines, including IL10, TGF-B, BDNF, and IGF, and increasing NSC proliferation, neural differentiation, and neural survival. BDNF, brain-derived neurotrophic factor; CCL2, C–C motif chemokine ligand 2; IFN-γ, interferon-gamma; IGF, insulin-like growth factor; IL-1β, interleukin 1 beta; IL-4, interleukin 4; IL-6, interleukin 6; IL-10, interleukin 10; IL-12, interleukin 12; NSC, neural stem cell; ROS, reactive oxygen species; TGF-β, transforming growth factor-betta; TNF-α, tumor necrosis factor-alpha; NeuN, neuronal nuclear protein
An overview of the intracellular mechanisms of action of soluble and transmembrane TNF-α via TNF receptors. TNF-α exerts its physiological and pathological effects by binding to TNFR-1 or TNFR-2, single transmembrane glycoproteins expressed by several cells. In this regard, TNF signaling through TNFR-1 plays a significant role in NPC proliferation, while TNFR-1 signaling inhibits NPC proliferation. TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2; FADD, Fas-associated death domain protein; MAPK, mitogen-activated protein kinase; MLKL, mixed-lineage kinase domain-like protein; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; RIP, receptor-interacting protein; PI3K: phosphatidylinositol 3-kinase; TRADD, TNF receptor-associated death domain protein; TRAF2, TNF receptor-associated factor 2; NPC, neural progenitor cell
Neuroinflammatory changes during the early and late phases of post-TBI period and effects on regeneration and neurogenesis. Neuroinflammation exists in acute and chronic TBI and plays a significant role in regulating post-TBI responses. In this regard, anti-inflammatory cytokines produced by M2 microglia following TBI result in higher synaptic plasticity, cell proliferation, and survival. While M1 microglia-produced cytokines increase neurodegeneration and decrease regeneration in post-TBI phases. TBI, traumatic brain injury; IL-1β, interleukin 1 beta; IL-4, interleukin 4; IL-6, interleukin 6; TGF-β, transforming growth factor-betta; TNF-α, tumor necrosis factor-alpha
Adult neurogenesis occurs mainly in the subgranular zone of the hippocampal dentate gyrus and the subventricular zone of the lateral ventricles. Evidence supports the critical role of adult neurogenesis in various conditions, including cognitive dysfunction, Alzheimer's disease (AD), and Parkinson's disease (PD). Several factors can alter adult neurogenesis, including genetic, epigenetic, age, physical activity, diet, sleep status, sex hormones, and central nervous system (CNS) disorders, exerting either pro-neurogenic or anti-neurogenic effects. Compelling evidence suggests that any insult or injury to the CNS, such as traumatic brain injury (TBI), infectious diseases, or neurodegenerative disorders, can provoke an inflammatory response in the CNS. This inflammation could either promote or inhibit neurogenesis, depending on various factors, such as chronicity and severity of the inflammation and underlying neurological disorders. Notably, neuroinflammation, driven by different immune components such as activated glia, cytokines, chemokines, and reactive oxygen species, can regulate every step of adult neurogenesis, including cell proliferation, differentiation, migration, survival of newborn neurons, maturation, synaptogenesis, and neuritogenesis. Therefore, this review aims to present recent findings regarding the effects of various components of the immune system on adult neurogenesis and to provide a better understanding of the role of neuroinflammation and neurogenesis in the context of neurological disorders, including AD, PD, ischemic stroke (IS), seizure/epilepsy, TBI, sleep deprivation, cognitive impairment, and anxiety- and depressive-like behaviors. For each disorder, some of the most recent therapeutic candidates, such as curcumin, ginseng, astragaloside, boswellic acids, andrographolide, caffeine, royal jelly, estrogen, metformin, and minocycline, have been discussed based on the available preclinical and clinical evidence.
 
Sphingosine receptors (S1PRs) are implicated in the progression of neurodegenerative diseases and metabolic disorders like obesity and type 2 diabetes (T2D). The link between S1PRs and cognition in type 2 diabetes, as well as the mechanisms that underpin it, are yet unknown. Neuroinflammation is the common pathology shared among T2D and cognitive impairment. However, the interplay between the M1 and M2 polarization state of microglia, a primary driver of neuroinflammation, could be the driving factor for impaired learning and memory in diabetes. In the present study, we investigated the effects of fingolimod (S1PR1 modulator) on cognition in high-fat diet and streptozotocin-induced diabetic mice. We further assessed the potential pathways linking microglial polarization and cognition in T2D. Fingolimod (0.5 mg/kg and 1 mg/kg) improved M2 polarization and synaptic plasticity while ameliorating cognitive decline and neuroinflammation. Sphingolipid dysregulation was mimicked in vitro using palmitate in BV2 cells, followed by conditioned media exposure to Neuro2A cells. Mechanistically, type 2 diabetes induced microglial activation, priming microglia towards the M1 phenotype. In the hippocampus and cortex of type 2 diabetic mice, there was a substantial drop in pSTAT3, which was reversed by fingolimod. This protective effect of fingolimod on microglial M2 polarization was primarily suppressed by selective jmjd3 blockade in vitro using GSK-J4, revealing that jmjd3 was involved downstream of STAT3 in the fingolimod-enabled shift of microglia from M1 to M2 polarization state. This study suggested that fingolimod might effectively improve cognition in type 2 diabetes by promoting M2 polarization.
 
Musashi RNA-binding proteins (MSIs) retain a pivotal role in stem cell maintenance, tumorigenesis, and nervous system development. Recently, we showed in C. elegans that Musashi (MSI-1) actively promotes forgetting upon associative learning via a 3’UTR-dependent translational expression of the Arp2/3 actin branching complex. Here, we investigated the evolutionary conserved role of MSI proteins and the effect of their pharmacological inhibition on memory. Expression of human Musashi 1 (MSI1) and Musashi 2 (MSI2) under the endogenous Musashi promoter fully rescued the phenotype of msi-1(lf) worms. Furthermore, pharmacological inhibition of human MSI1 and MSI2 activity using (-)- gossypol resulted in improved memory retention, without causing locomotor, chemotactic, or learning deficits. No drug effect was observed in msi-1(lf) treated worms. Using Western blotting and confocal microscopy, we found no changes in MSI-1 protein abundance following (-)- gossypol treatment, suggesting that Musashi gene expression remains unaltered and that the compound exerts its inhibitory effect post-translationally. Additionally, (-)- gossypol suppressed the previously seen rescue of the msi-1(lf) phenotype in worms expressing human MSI1 specifically in the AVA neuron, indicating that (-)- gossypol can regulate the Musashi pathway in a memory-related neuronal circuit in worms. Finally, treating aged worms with (-)- gossypol reversed physiological age-dependent memory decline. Taken together, our findings indicate that pharmacological inhibition of Musashi might represent a promising approach for memory modulation.
 
Transient receptor potential melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation channel that plays a key role in cell functions such as ion homeostasis, cell proliferation, survival, and cytoskeletal dynamics and mediates cells death in hypoxic and ischemic conditions. Previously, TRPM7 was found to play a role in the neurite outgrowth and maturation of primary hippocampal neurons. Either knockdown of TRPM7 with target-specific shRNA or blocking channel conductance by a specific blocker waixenicin A enhanced axonal outgrowth in the primary neuronal culture. In this study, we investigated whether and how TPRM7 is involved in hypoxia-altered neurite outgrowth patterns in E16 hippocampal neuron cultures. We demonstrate that short-term hypoxia activated the MEK/ERK and PI3K/Akt pathways, reduced TRPM7 activity, and enhanced axonal outgrowth of neuronal cultures. On the other hand, long-term hypoxia caused a progressive retraction of axons and dendrites that could be attenuated by the TRPM7-specific inhibitor waixenicin A. Further, we demonstrate that in the presence of astrocytes, axonal retraction in long-term hypoxic conditions was enhanced, and TRPM7 block by waixenicin A prevented this retraction. Our data demonstrate the effect of hypoxia on TRPM7 activity and axonal outgrowth/retraction in cultures with or without astrocytes present.
 
CORM-A1 induces mitophagy in astrocytes. A Primary culture of astrocytes isolated from mitoQC transgenic pups was pre-treated with 12.5 µM of CORM-A1 for 1 h, 3 h, 6 h, 8 h, and 24 h. Representative picture of astrocyte mitophagy followed by fluorescent microscopy. Mitochondrial protein FIS1 is coupled to GFP and RFP. When mitochondrial are engulfed by lysosomes for degradation, the green signal of the GFP is quenched inside de acidic lumen of the lysosome and only the red signal is visible. Thus, red dots are indicative of mitolysosomes (mitophagy). B Quantification of mitolysosomes per cell; graphs represent the mean ± SEM of 3 biological experiments; data were analysed with unpaired t-test; *p < 0.05 and **p < 0.01. C Primary cultures of astrocytes cultured in 24-well plate were treated with 25 µM CCCP or 12.5 µM of CORM-A1 for 1 h with and without the inhibitor CsA at 5 µM, which was added simultaneously with mitophagy inducers. MTDR was used to determine mitochondrial mass by flow cytometry. The intensity in the FL4 channel was normalized to untreated control cells. Graphs represent the mean ± SEM of five experiments performed in triplicate, analysed with one way ANOVA test. *p < 0.05 and **p < 0.01 compared with control and ##p < 0.01 compared to CORM-A1 treatment. D In order to control the role of CO in the modulation of mitochondrial population and mitophagy, MTDR-based mitochondrial population was performed in the presence of 12.5 µM of inactivated CORM-A1 or 50 µM of CO gas (PBS-saturated solution) for 1 h and 24 h. Graphs represent the mean ± SEM of five experiments performed in triplicate, analysed with the two-way ANOVA test. *p < 0.05 compared to control
CORM-A1-induces PINK1/Parkin-dependent mitophagy in astrocytes. A Mitochondrial fractions isolated from primary culture of astrocytes treated with CORM-A1 at a final concentration of 12.5 µM or with CCCP at 25 µM (positive control) during 1 h were analysed by immunoblotting using antibodies against PINK1 and Parkin and normalized using anti-COX11. B, C Quantification of the presence of PINK1 (B) and Parkin (C) in mitochondrial enriched fraction. Graphs represent the mean ± SEM of four experiments analysed with Mann–Whitney test, with *p < 0.05. D Primary culture of astrocytes isolated from mitoQC transgenic animals and with PINK1 expression knocked down with siRNA (10 pmol) was pre-treated with 12.5 µM of CORM-A1 for 1 h. Representative picture of astrocyte mitophagy assessed fluorescent microscopy. E Quantification of mitolysosomes (red dots) per cell (number of cells per condition ≥ 20). Graphs represents mean ± SEM. Statistic was made using two-way ANOVA test with *p < 0.05 and **p < 0.01 compared to control without treatment, and ##p < 0.01 compared to CORM-A1 treatment
CORM-A1 induces mitochondrial biogenesis. A Primary culture of astrocytes isolated from mitoQC transgenic mice pups was pre-treated with 12.5 µM of CORM-A1 for 24 h. Representative picture of astrocytic mitophagy followed by fluorescent microscopy. B Quantification of mitolysosomes (red dots) per cell. Graph represents the mean ± SEM of 3 experiments performed with 4 technical replicas, and analysed with unpaired t-test with *p < 0.05. C Primary cultures of astrocytes cultured in 24-well plate were treated with 25 µM CCCP or 12.5 µM of CORM-A1 for 24 h, with and without the mitophagy inhibitor CsA at 5 µM or the protein synthesis inhibitor chloramphenicol (Cm) at 10 µM; both inhibitors were added simultaneously with mitophagy inducers. MTDR was used to determine mitochondrial mass by flow cytometry. The intensity in the FL4 channel was normalized to untreated control cells. Graphs represent the mean ± SEM of five experiments performed in triplicate, analysed with the one-way ANOVA test. D Primary cultures of astrocytes cultured in 24-well plate were treated with 12.5 µM of CORM-A1 and 25 µM of CCCP for 1 h and 24 h, followed by DNA extraction for measuring mitochondrial cytochrome b (mtCyt b) gene to assess mitochondrial DNA amount, which is represented by fold increase when compared to control. Graphs represent the mean ± SEM of four experiments performed in triplicate; data were analysed with two-way ANOVA test. E Primary cultures of astrocytes cultured in 24-well plate were treated with 12.5 µM of CORM-A1 for 1 h and 24 h, followed by mRNA extraction for measuring mitochondrial PGC-1α gene to assess mitochondrial biogenesis, which is represented by fold increase when compared with control. All values are mean ± SEM of five experiments performed in triplicate and data were analysed with Bonferroni’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with control
CORM-A1-induced mitochondrial biogenesis and CORM-A1 cytoprotection are dependent on mitophagy. A Primary cultures of astrocytes cultured in 24-well plate were treated with PINK1 siRNA for knocking down its expression. After 24 h, astrocytes were treated with 12.5 µM of CORM-A1 for 1 h, followed by mRNA extraction for measuring mitochondrial PGC-1α gene to assess mitochondrial biogenesis, which is represented by fold increase when compared with control. All values are mean ± SEM (n = 5) and data were analysed with the Mann–Whitney test. **p < 0.01 and #p < 0.05 compared with CORM-A1 treatment. B Cell viability was assessed by flow cytometry, using propidium iodide that dyes permeable plasma membrane, which are considered dead cells. Astrocytic PINK1 expression was knocked down for 24 h; then, astrocytes were treated with 12.5 µM of CORM-A1 for 24 h, followed by challenging them to die with the pro-oxidant t-BHP (0 to 320 µm) for 18 h. All the values are mean ± SEM (n = 3) and data were analysed with the Mann–Whitney test. *p < 0.05 compared to control and #p < 0.05 compared with CORM-A1 treatment
Astrocytes are key glial cells for the metabolic and functional support of the brain. Mitochondrial quality control (MQC), in particular the balance between mitophagy and mitochondrial biogenesis, is a major event for the maintenance of cellular homeostasis. Carbon monoxide (CO) is an endogenous gasotransmitter that inhibits cell death and inflammation by targeting mitochondria. It is well established that CO promotes cytoprotection by increasing mitochondrial population and metabolism (oxidative phosphorylation). Thus, it is hypothesized that CO-induced cytoprotection may also be mediated by the balance between mitophagy and mitochondrial biogenesis. Herein, the carbon monoxide releasing molecule-A1 (CORM-A1) was used in primary cultures of astrocytes to assess CO role on mitochondrial turnover. PINK1/Parkin-dependent mitophagy was stimulated by CORM-A1 following 1 h of treatment. While at 24 h after treatment, CORM-A1 increased mitochondrial population, which may indicate mitochondrial biogenesis. In fact, mitochondrial biogenesis was confirmed by the enhancement of PGC-1α expression that upregulates several mitochondrial transcription factors. Furthermore, inhibition of mitophagy by knocking down PINK1 expression reverted CO-induced mitochondrial biogenesis, indicating that mitochondrial turnover is dependent on modulation of mitophagy. Finally, CORM-A1 prevented astrocytic cell death induced by oxidative stress in a mitophagy-dependent manner. In fact, whenever PINK1 was knocked down, CORM-A1-induced cytoprotection was lost. In summary, CORM-A1 stimulates mitochondrial turnover, which in turn prevents astrocytic cell death. CO cytoprotection depends on increasing mitochondrial population and on eliminating dysfunctional mitochondria.
 
The therapeutic application of neural stem cells (NSCs) in the central nerve system (CNS) injury is a promising strategy for combating irreversible neuronal loss. However, a variety of obvious inflammatory responses following nerve injury rapidly create an unfavorable microenvironment for survival and neuronal differentiation of NSCs in lesion area, limiting the efficacy of NSC-based therapy for CNS injury. It remained unknown how to effectively increase the neuronal differentiation efficiency of NSCs through transplantation. Here, we demonstrated that curcumin (CCM)-activated olfactory ensheathing cells (aOECs) effectively promoted neuronal differentiation of NSCs in the activated microglial inflammatory condition, and co-transplantation of aOECs and NSCs improved neurological recovery of rats after spinal cord injury (SCI), as evidenced by higher expression levels of neuronal markers and lower expression levels of glial markers in the differentiated cells, greater number of Tuj-1-positive cells as well as higher Basso, Beattie, and Bresnahan (BBB) locomotor scale, compared to the corresponding controls. Pathologically, hematoxylin and eosin (HE) staining and immunostaining also showed that aOECs remarkably enhanced the in vivo neuronal differentiation of NSCs and migration, and nerve repair. Further analysis revealed that the underlying mechanisms of aOECs potentiating the neuronal conversion of NSCs under inflammatory environment were tightly associated with up-regulation of anti-inflammatory cytokines and neurotrophic factors in OECs, and importantly, the activation of Wnt3/β-catenin pathway was likely involved in the mechanisms underlying the observed cellular events. Therefore, this study provides a promising strategy for SCI repair by co-transplantation of aOECs and NSCs.
 
Therapeutic hypothermia (TH) is the only intervention approved for the treatment of neonatal hypoxic-ischaemic encephalopathy (HIE), but its treatment window is narrow (within 6 h after birth), and its efficacy is not ideal. Thus, alternative treatments are urgently needed. Our previous studies showed that genistein-3′-sodium sulfonate (GSS), a derivative of genistein (Gen), has a strong neuroprotective effect in rats with ischaemic stroke, but its role in HIE is unclear. A hypoxia–ischaemia (HI) brain injury model was established in neonatal male Sprague‒Dawley (SD) rats. Twenty-four hours after reperfusion, rats treated with GSS were assessed for cerebral infarction, neurological function, and neuronal damage. RNA-Seq and bioinformatics analysis were used to explore differentially expressed genes (DEGs) and regulated signalling pathways, which were subsequently validated by Western blotting and immunofluorescence. In this study, we found that GSS not only significantly reduced the size of brain infarcts and alleviated nerve damage in rats with HIE but also inhibited neuronal loss and degeneration in neonatal rats with HIE. A total of 2170 DEGs, of which 1102 were upregulated and 1068 were downregulated, were identified in the GSS group compared with the HI group. In an analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) categories, the downregulated DEGs were significantly enriched in the pathways “Phagosome”, “NF-κB signalling”, and “Complement and coagulation cascades”, amongst others. Meanwhile, the upregulated DEGs were significantly enriched in the pathways “Neurodegeneration”, “Glutamatergic synapse”, and “Calcium signalling pathway”, amongst others. These results indicate that GSS intervenes in the process of HIE-induced brain injury by participating in multiple pathways, which suggests potential candidate drugs for the treatment of HIE. Graphical Abstract
 
Parkinson’s disease (PD) is the second most common neurodegenerative disorder and is caused by the loss of dopaminergic neurons in the substantia nigra (SN). However, the reason for the death of dopaminergic neurons remains unclear. An increase in α-synuclein (α-syn) expression is an important factor in the pathogenesis of PD. In the current study, we investigated the association between serine/arginine-rich protein-specific kinase 3 (Srpk3) and PD in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in SH-SY5Y cells treated with 1-methyl-4-phenylpyridinium (MPP+). Srpk3 expression was significantly downregulated, while tyrosine hydroxylase (TH) expression decreased and α-syn expression increased after 4 weeks of MPTP treatment. Dopaminergic cell reduction and α-syn expression increase were demonstrated by Srpk3 expression inhibition by siRNA in SH-SY5Y cells. Moreover, a decrease in Srpk3 expression upon siRNA treatment promoted dopaminergic cell reduction and α-syn expression increase in SH-SY5Y cells treated with MPP+ . These results suggested that Srpk3 expression decrease due to Srpk3 siRNA caused both TH level decrease and α-syn expression increase. This raises new possibilities for studying how Srpk3 controls dopaminergic cells and α-syn expression, which may be related to PD pathogenesis. Our results provide an avenue for understanding the role of Srpk3 in dopaminergic cell loss and α-syn upregulation in SN. Furthermore, this study supports a therapeutic possibility for PD in that the maintenance of Srpk3 expression inhibits dopaminergic cell reduction.
 
Normalized fold mRNA expression of inflammation markers, such as a glial fibrillary acidic protein (Gfap); b allograft inflammatory factor 1 (Aif1); c interleukin-1 beta (Il1b) in the brain prefrontal cortices of 5xFAD mice (n = 7) and wild-type (WT) control mice (n = 6). The normalization of gene expression was performed against the housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh). The data are expressed as mean ± SD. Asterisks denote a statistically significant difference from the respective control (***p < 0.005, **** < 0.001, unpaired t-test)
Comparison of absolute protein expression levels of the transporters in the crude membrane fraction of a isolated brain microvessels of TgF344-AD rats (n = 6) [28] versus 5xFAD mice from the present study (n = 6); b isolated brain microvessels of WT rats (n = 6) [28] versus 8-month-old WT mice from the present study (n = 6); c the brain cortices of TgF344-AD rats (n = 8) [28] versus 5xFAD mice from the present study (n = 12); d the brain cortices of WT rats (n = 5) [28] versus 8-month-old WT mice from the present study (n = 14); e the brain cortices of female APdE9 mice (n = 4) [29] versus female 5xFAD mice from the present study (n = 12); f the brain cortices of female 8-month-old WT mice (n = 5) from the present study versus female 16–17-month-old WT mice (n = 14) [29]. The top and bottom dashed lines represent a twofold higher or lower protein expression, respectively, between the studied groups. Data are expressed as mean ± SD
a Relative comparison of amino acid concentrations in the brain prefrontal cortices of 5xFAD mice (n = 9) and WT controls (n = 9). b Relative comparison of amino acid concentrations in the plasma of 5xFAD mice (n = 9) and WT controls (n = 9). The data are expressed as mean ± SD. Asterisks denote a statistically significant difference from the respective control (* < 0.05, ** < 0.01, ***p < 0.005, unpaired t-test)
Membrane transporters such as ATP-binding cassette (ABC) and solute carrier (SLC) transporters expressed at the neurovascular unit (NVU) play an important role in drug delivery to the brain and have been demonstrated to be involved in Alzheimer’s disease (AD) pathogenesis. However, our knowledge of quantitative changes in transporter absolute protein expression and functionality in vivo in NVU in AD patients and animal models is limited. The study aim was to investigate alterations in protein expression of ABC and SLC transporters in the isolated brain microvessels and brain prefrontal cortices of a widely used model of familial AD, 5xFAD mice (8 months old), using a sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic approach. Moreover, we examined alterations in brain prefrontal cortical and plasmatic levels of transporter substrates in 5xFAD mice compared to age-matched wild-type (WT) controls. ASCT1 (encoded by Slc1a4) protein expression in the isolated brain microvessels and brain prefrontal cortices of 5xFAD mice was twice higher compared to WT controls (p = 0.01). Brain cortical levels of ASCT1 substrate, serine, were increased in 5xFAD mice compared to WT animals. LAT1 (encoded by Slc7a5) and 4F2hc (encoded by Slc3a2) protein expressions were significantly altered in the isolated brain microvessels of 5xFAD mice compared to WT controls (p = 0.008 and p = 0.05, respectively). Overall, the study provides important information, which is crucial for the optimal use of the 5xFAD mouse model in AD drug development and for investigating novel drug delivery approaches. In addition, the findings of the study shed light on the novel potential mechanisms underlying AD pathogenesis.
 
Chromosome topology at the level of chromatin loops (a), TADs (b) and A/B compartments (c). Deletion of CTCF binding sites can lead to formation of new TADs, which can cause genes to contact enhancers normally spatially too far away to do so (a process known as “enhancer hijacking”) (d). These new enhancer-promoter contacts can cause ectopic gene expression patterns that have pathological consequences
Flow diagram showing numbers of studies found and fully reviewed or excluded
Abnormal chromatin folding perturbs normal enhancer-promoter contacts and subsequent transcriptional patterns. Mutations and genetic structural modifications converge on mechanisms perturbing normal enhancer-promoter contact formation, including TAD re-organisation, altered chromatin accessibility and mutations at the enhancer or promoter DNA sequences preventing their normal association. These general mechanisms then pathologically converge on abnormal transcriptional outputs (including excessive or no transcriptional activity)
How DNA is folded and packaged in nucleosomes is an essential regulator of gene expression. Abnormal patterns of chromatin folding are implicated in a wide range of diseases and disorders, including epilepsy and autism spectrum disorder (ASD). These disorders are thought to have a shared pathogenesis involving an imbalance in the number of excitatory-inhibitory neurons formed during neurodevelopment; however, the underlying pathological mechanism behind this imbalance is poorly understood. Studies are increasingly implicating abnormal chromatin folding in neural stem cells as one of the candidate pathological mechanisms, but no review has yet attempted to summarise the knowledge in this field. This meta-synthesis is a systematic search of all the articles on epilepsy, ASD, and chromatin folding. Its two main objectives were to determine to what extent abnormal chromatin folding is implicated in the pathogenesis of epilepsy and ASD, and secondly how abnormal chromatin folding leads to pathological disease processes. This search produced 22 relevant articles, which together strongly implicate abnormal chromatin folding in the pathogenesis of epilepsy and ASD. A range of mutations and chromosomal structural abnormalities lead to this effect, including single nucleotide polymorphisms, copy number variants, translocations and mutations in chromatin modifying. However, knowledge is much more limited into how abnormal chromatin organisation subsequently causes pathological disease processes, not yet showing, for example, whether it leads to abnormal excitation-inhibitory neuron imbalance in human brain organoids.
 
The lateral hypothalamus (LH) has a heterogeneous cytoarchitectonic organization that has not been elucidated in detail. In this work, we analyzed within the framework of the prosomeric model the differential expression pattern of 59 molecular markers along the ventrodorsal dimension of the medial forebrain bundle in the mouse, considering basal and alar plate subregions of the LH. We found five basal (LH1–LH5) and four alar (LH6–LH9) molecularly distinct sectors of the LH with neuronal cell groups that correlate in topography with previously postulated alar and basal hypothalamic progenitor domains. Most peptidergic populations were restricted to one of these LH sectors though some may have dispersed into a neighboring sector. For instance, histaminergic Hdc-positive neurons were mostly contained within the basal LH3, Nts (neurotensin)- and Tac2 (tachykinin 2)-expressing cells lie strictly within LH4, Hcrt (hypocretin/orexin)-positive and Pmch (pro-melanin-concentrating hormone)-positive neurons appeared within separate LH5 subdivisions, Pnoc (prepronociceptin)-expressing cells were mainly restricted to LH6, and Sst (somatostatin)-positive cells were identified within the LH7 sector. The alar LH9 sector, a component of the Foxg1-positive telencephalo-opto-hypothalamic border region, selectively contained Satb2-expressing cells. Published studies of rodent LH subdivisions have not described the observed pattern. Our genoarchitectonic map should aid in systematic approaches to elucidate LH connectivity and function.
 
Coumarins are plant-derived polyphenolic compounds belonging to the benzopyrones family, possessing wide-ranging pharmaceutical applications including cytoprotection, which may translate into therapeutic potential for multiple diseases, including Parkinson’s disease (PD). Here we demonstrate the neuroprotective potential of a new polyhydroxyl coumarin, N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamide (CT51), against the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+). MPP+’s mechanism of toxicity relates to its ability to inhibit complex I of the mitochondrial electron transport chain (METC), leading to adenosine triphosphate (ATP) depletion, increased reactive oxygen species (ROS) production, and apoptotic cell death, hence mimicking PD-related neuropathology. Dopaminergic differentiated human neuroblastoma cells were briefly pretreated with CT51, followed by toxin exposure. CT51 significantly restored somatic cell viability and neurite processes; hence, the drug targets cell bodies and axons thereby preserving neural function and circuitry against PD-related damage. Moreover, MPP+ emulates the iron dyshomeostasis affecting dopaminergic neurons in PD-affected brains, whilst CT51 was previously revealed as an effective iron chelator that preferentially partitions to mitochondria. We extend these findings by characterising the drug’s interactive effects at the METC level. CT51 did not improve mitochondrial coupling efficiency. However, voltammetric measurements and high-resolution respirometry analysis revealed that CT51 acts as an antioxidant agent. Also, the neuronal protection afforded by CT51 associated with downregulating MPP+-induced upregulated expression of hypoxia-inducible factor 1 alpha (HIF-1α), a protein which regulates iron homeostasis and protects against certain forms of oxidative stress after translocating to mitochondria. Our findings support the further development of CT51 as a dual functioning iron chelator and antioxidant antiparkinsonian agent.
 
Hypoglycemia is associated with cognitive dysfunction, but the exact mechanisms have not been elucidated. Our previous study found that severe hypoglycemia could lead to cognitive dysfunction in a type 1 diabetes (T1D) mouse model. Thus, the aim of this study was to further investigate whether the mechanism of severe hypoglycemia leading to cognitive dysfunction is related to oxidative stress-mediated pericyte loss and blood-brain barrier (BBB) leakage. A streptozotocin T1D model (150 mg/kg, one-time intraperitoneal injection), using male C57BL/6J mice, was used to induce hypoglycemia. Brain tissue was extracted to examine for neuronal damage, permeability of BBB was investigated through Evans blue staining and electron microscopy, reactive oxygen species and adenosine triphosphate in brain tissue were assayed, and the functional changes of pericytes were determined. Cognitive function was tested using Morris water maze. Also, an in vitro glucose deprivation model was constructed. The results showed that BBB leakage after hypoglycemia is associated with excessive activation of oxidative stress and mitochondrial dysfunction due to glucose deprivation/reperfusion. Interventions using the mitochondria-targeted antioxidant Mito-TEMPO in both in vivo and in vitro models reduced mitochondrial oxidative stress, decreased pericyte loss and apoptosis, and attenuated BBB leakage and neuronal damage, ultimately leading to improved cognitive function.
 
tRFs biogenesis from pre and mature tRNA
Types of tRFs involved in various neurological disorders
Role of tRFs derived from 5′end in neurological disorders. (1) In HD samples, upregulation of tRNA-Ala-CGC-3 cause reduction in cell viability of certain neurons. (2) In epilepsy samples, tRF5s, 5′AlaTGC, 5′GlyGCC, and 5′GluCTC are highly secreted from neuron prior to seizure but it reduces post seizure. (3) In TBI, unregulated tRF-Gly-GCC-017 leads to reduction in synaptic function and downregulation of tRF-Thr-AGT-003 and tRF-Ser-GCT-078 results in increased inflammation. (4) The downregulation of tRF-Leu-CAA-004 results in increased efflux of calcium to plasma and affects retinol metabolism in AD samples. A tRF-5, AS-tDR-013428 was downregulated, involved in amyloid beta peptide production leading to neurotoxicity. Expression of tRF5-GlyGCC, tRF5-GluCTC, and tRF5-GlyCCC-2 were more in EOAD and expression of tRF5-Pro-AGG was dependent on age and stage. Upregulation of tRF5-Cys-GCA leads to reduced atrophy. (5) In stroke samples, downregulated rno-tRF5-Ala-16a and rno-tRF5-Glu-29a are involved in regulation of embryonic morphogenesis, permeability of BBB and inflammation. rno-tRF5-Glu-29a also deals with regulation of G protein coupled receptor pathway and cytoskeleton organization. (6) Downregulated mt 5′-tRF LeuUUR regulates expression of OXPHOS complex 1 subunit. Upregulated mt 5′-tRF LeuUUR-m.3243A > G exhibited similar expression pattern in cybrid cells, fibroblast, and biofluid samples
tRFs are small tRNA derived fragments that are emerging as novel therapeutic targets and regulatory molecules in the pathophysiology of various neurological disorders. These are derived from precursor or mature tRNA, forming different subtypes that have been reported to be involved in neurological disorders like stroke, Alzheimer’s, epilepsy, Parkinson’s, MELAS, autism, and Huntington’s disorder. tRFs were earlier believed to be random degradation debris of tRNAs. The significant variation in the expression level of tRFs in disease conditions indicates their salient role as key players in regulation of these disorders. Various animal studies are being carried out to decipher their exact role; however, more inputs are required to transform this research knowledge into clinical application. Future investigations also call for high-throughput technologies that could help to bring out the other hidden aspects of these entities. However, studies on tRFs require further research efforts to overcome the challenges posed in quantifying tRFs, their interactions with other molecules, and the exact mechanism of function. In this review, we are abridging the current understanding of tRFs, including their biogenesis, function, relevance in clinical therapies, and potential as diagnostic and prognostic biomarkers of these neurological disorders.
 
The mechanisms of treatment-resistant depression (TRD) are not clear and are difficult to study. An animal model resembling human TRD is the Wistar Kyoto rat strain. In the present study, we focused on selecting miRNAs that differentiate rats of the WKY strain from Wistar Han (WIS) rats in two divisions of the habenula, the lateral and medial (LHb and MHb, respectively). Based on our preliminary study and literature survey, we identified 32 miRNAs that could be potentially regulated in the habenula. Six miRNAs significantly differentiated WKY rats from WIS rats within the MHb, and three significantly differentiated WKY from WIS rats within the LHb. Then, we selected relevant transcripts regulated by those miRNAs, and their expression in the habenular nuclei was investigated. For mRNAs that differentiated WKY rats from WIS rats in the MHb (Cdkn1c, Htr7, Kcnj9, and Slc12a5), their lower expression correlated with a higher level of relevant miRNAs. In the LHb, eight mRNAs significantly differentiated WKY from WIS rats (upregulated Htr4, Drd2, Kcnj5, and Sstr4 and downregulated Htr2a, Htr7, Elk4, and Slc12a5). These data indicate that several important miRNAs are expressed in the habenula, which differentiates WKY rats from WIS rats and in turn correlates with alterations in the expression of target transcripts. Of particular note are two genes whose expression is altered in WKY rats in both LHb and MHb: Slc12a5 and Htr7. Regulation of KCC2 via the 5-HT7 receptor may be a potential target for the treatment of TRD.
 
Substantial evidence suggests that pyroptosis is involved in renal, cerebral, and myocardial ischemia–reperfusion injury. However, whether pyroptosis is involved in ischemia–reperfusion injury of cochlear hair cells has not been explored. In this study, we examined the effects of melatonin on the oxygen–glucose deprivation/reperfusion (OGD/R) of hair cell-like House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear hair cells in vitro to mimic cochlear ischemia–reperfusion injury in vivo. We found that melatonin treatment protected the HEI-OC1 and cochlear hair cells against OGD/R-induced cell pyroptosis and reduced the expression level of ROS in these cells. However, these effects were completely abolished by the application of luzindole (a non-selective melatonin receptor blocker) and largely offset by the use of ML385 (an nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor). These findings suggest that melatonin alleviates OGD/R-induced pyroptosis of the hair cell-like HEI-OC1 cells and cochlear hair cells via the melatonin receptor 1A (MT-1) and melatonin receptor 1B (MT-2)/Nrf2 (NFE2L2)/ROS/NLRP3 pathway, which may provide credible evidence for melatonin being used as a potential drug for the treatment of idiopathic sudden sensorineural hearing loss in the future.
 
The peri- and post-menopausal periods have been described as the “window of vulnerability” for the development of depressive symptoms that impair women activities and quality of life. The etiopathogenesis of these symptoms is multifactorial and may confer resistance to traditional antidepressants. Attention is now directed toward phytochemicals for their pleiotropic functions and safer profiles. This study investigated the possible perturbation of the nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathways as an underlying mechanism of post-ovariectomy depression and highlighted the potential benefits of carnosic acid (CA) on the associated behavioral, biochemical, and histopathological alterations. Female Balb/c mice were randomly assigned to be sham-operated or ovariectomized (OVX). After 3 weeks, OVX mice received either a vehicle, CA (20 mg/kg/day), or tin protoporphyrin IX (SnPP-IX; a heme oxygenase-1 (HO-1) inhibitor; 50 μmol/kg/day) for 3 weeks. Our findings revealed that OVX mice had depressive but not anxiety-like behavior. Suppressed Nrf2 and its downstream signaling, and augmented proinflammatory markers were observed in both the hippocampus and prefrontal cortex. CA treatment alleviated depressive behavior, induced the expression of Nrf2, HO-1, thioredoxin-1, and brain-derived neurotrophic factor, and enhanced serotonin levels. CA also suppressed oxidative stress, reduced TNF-α, IL-1β, and iNOS mRNA expression, and ameliorated OVX-induced histopathological changes. SnPP-IX aggravated post-OVX behavioral, neurobiochemical, and histological deteriorations, and reduced CA-protective effects. In conclusion, Nrf2/HO-1 signaling suppression and the associated proinflammatory state are key mechanisms in post-OVX depression. CA exerts multifaceted neuroprotection in OVX mice and represents a promising candidate for clinical evaluation as an antidepressant.
 
Amorfrutin B is a selective modulator of the PPARγ receptor, which has recently been identified as an effective neuroprotective compound that protects brain neurons from hypoxic and ischemic damage. Our study demonstrated for the first time that a 6-h delayed post-treatment with amorfrutin B prevented hypoxia/ischemia-induced neuronal apoptosis in terms of the loss of mitochondrial membrane potential, heterochromatin foci formation, and expression of specific genes and proteins. The expression of all studied apoptosis-related factors was decreased in response to amorfrutin B, both during hypoxia and ischemia, except for the expression of anti-apoptotic BCL2, which was increased. After post-treatment with amorfrutin B, the methylation rate of the pro-apoptotic Bax gene was inversely correlated with the protein level, which explained the decrease in the BAX/BCL2 ratio as a result of Bax hypermethylation. The mechanisms of the protective action of amorfrutin B also involved the inhibition of autophagy, as evidenced by diminished autophagolysosome formation and the loss of neuroprotective properties of amorfrutin B after the silencing of Becn1 and/or Atg7. Although post-treatment with amorfrutin B reduced the expression levels of Becn1, Nup62, and Ambra1 during hypoxia, it stimulated Atg5 and the protein levels of MAP1LC3B and AMBRA1 during ischemia, supporting the ambiguous role of autophagy in the development of brain pathologies. Furthermore, amorfrutin B affected the expression levels of apoptosis-focused and autophagy-related miRNAs, and many of these miRNAs were oppositely regulated by amorfrutin B and hypoxia/ischemia. The results strongly support the position of amorfrutin B among the most promising anti-stroke and wide-window therapeutics.
 
Heat map of 12 upregulated and 15 downregulated lncRNAs in the pilot study (all Q < 0.05, with FDR correction, dataset 1). Red and blue indicate upregulation and downregulation, respectively. Abbreviations: FDR, false discovery rate; AD, Alzheimer’s disease
Measurements of lncRNAs in dataset 2. Changes in AD of LINC02067 (A), LINC00987 (B), LOC107987206 (C), ANKRD34C-AS1 (D), NORAD (E), THCAT158 (F), LOC105378179 (G), SLMO2-ATP5E (H), LOC105372826 (I), LOC101927647 (J), MALAT1 (K), and CCDC183-AS1 (L) were confirmed. Abbreviations: AD, Alzheimer’s disease; FC, fold change
Establishment of diagnostic panel for AD. (A) ROC curve analysis of the seven-lncRNA panel (upregulated: LINC02067, LINC00987, NORAD, ANKRD34C-AS1, THCAT158; downregulated: LOC107987206, LOC105378179). (B) ROC analysis of seven individual lncRNAs. Abbreviations: AD, Alzheimer’s disease; ROC, receiver operating characteristic; AUC, area under the curve
Measurements of lncRNAs in control, AD, VaD, PDD, bvFTD, and DLB. LINC02067 (A), LINC00987 (B), LOC107987206 (C), ANKRD34C-AS1 (D), NORAD (E), THCAT158 (F), and LOC105378179 (G) were measured. Abbreviations: AD, Alzheimer’s disease; VaD, vascular dementia; PDD, Parkinson’s disease dementia; bvFTD, behavioral variant frontotemporal dementia; DLB, dementia with Lewy body; FC, fold change
ROC curve analysis in dataset 3. The ROCs of AD versus controls (A), AD versus non-AD (B), and AD versus other types of dementia (C). Non-AD indicates a combination of controls and other types of dementia. Other types of dementia include VaD, PDD, bvFTD, and DLB. Abbreviations: ROC, receiver operating characteristic; AD, Alzheimer’s disease; VaD, vascular dementia; PDD, Parkinson’s disease dementia; bvFTD, behavioral variant frontotemporal dementia; DLB, dementia with Lewy body; AUC, area under the curve. *** P < 0.001
Long non-coding RNAs (lncRNAs) have been identified to be involved in the pathogenesis of Alzheimer’s disease (AD). In this study, we evaluated whether lncRNAs can be used to discriminate AD patients from controls and patients with other dementias, such as vascular, Parkinson’s disease, behavioral variant frontotemporal, and dementia with Lewy body. In this study, we used three datasets to measure the blood lncRNA levels. A pilot study (dataset 1, n = 40; controls, 20; AD, 20) was used to screen for differentially expressed lncRNAs. Dataset 2 (n = 174; controls, 86; AD, 88) was used to identify a lncRNA panel for the diagnostic model. Dataset 3 (n = 333; control, 60; AD, 54; vascular dementia, 53; Parkinson’s disease dementia, 55; behavioral variant frontotemporal dementia, 56; and dementia with Lewy body, 55) was used to validate the diagnostic model. In dataset 1, 12 upregulated and 15 downregulated lncRNAs were identified. In dataset 2, a panel of seven lncRNAs was found to have the ability to differentiate AD patients from controls. Finally, this panel was applied to dataset 3 to successfully distinguish AD from other dementias. This study proposes a panel of seven lncRNAs as specific and promising biomarker for AD diagnosis.
 
Acrylamide (ACR), a soft electrophile, is a typical environmental and food contaminant that presents potential health hazards and, consequently, is attracting increasing attention in the quest for its control. ACR neurotoxicity has been widely reported in experimental animals and attributed to neuroinflammation; however, the mechanisms involved therein require clarification. In this study, we used a neuron cell model to investigate the mechanisms of ACR-induced neuroinflammation and pyroptosis. The results showed that ACR treatment induced lytic cell death morphologically under both the canonical pyroptotic pathway (NOD-like receptor protein 3 (NLRP3)-apoptosis-associated speck-like protein containing CARD (ASC)-cysteinyl aspartate specific proteinase 1 (caspase-1)-gasdermin D (GSDMD)-interleukin-1β (IL-1β)/interleukin-18 (IL-18)) and an alternative pyroptotic pathway (cysteinyl aspartate specific proteinase 3 (caspase-3)-gasdermin E (GSDME)-IL-1β/IL-18) in SH-SY5Y cells. Moreover, the lactate dehydrogenase (LDH) production, cytokines release, and lytic cell death induced by ACR were diminished by caspase-1 and -3 inhibitors. Furthermore, the knockdown of caspase-1 by small interfering RNA attenuated ACR-induced lytic cell death, suggesting that canonical pyroptosis (the NLRP3-caspase 1-GSDMD-IL-1β signaling axis) played a primary role in the ACR-induced pyroptosis. Of the two pyroptotic-related pathways, the NLRP3 inflammasome cascade was activated first within the 6-h period of ACR exposure, while the activation of the alternative pyroptotic pathway was delayed. Collectively, these results indicate that ACR mainly induces NLRP3-related neuroinflammation and pyroptosis in SH-SY5Y cells, which is, thus, suggestive of an alternative mechanism for ACR-induced neurotoxicity.
 
Selective cilia deletion in the striatum and confirmation of mice’s normal gross growth and well-being. a Schematic view of experimental design and behavior assays performed and their sequence. Diagram was created with the BioRender.com webpage. b Schematic showing bilateral viral injection into the dorsal striatum. c and d Verification of cilia removal in ciliated neurons of the striatum using immunostaining of ADCY3. Scale bar = 10 μm. c Representative images of ADCY3 immunostaining showing the intact cilia in the control mice and the conditional ablation of cilia in the dorsal striatum neurons of Ift88fl mice (counterstained with DAPI, blue); d Quantification of the ciliated cells in the rostral-dorsal striatum (n = 8 control, 6 IFT88-KO). Unpaired t-test (t = 17.26, P < 0.0001) ****P < 0.0001. Data are presented as means ± S.E.M. Scale bar = 10 μm. e–g ADCY3 immunostaining in the caudal striatum. e Representative images of ADCY3 immunostaining in the caudal striatum showing that the selective removal of cilia from the dorsal rostral striatum does not affect f the number of ciliated cells (t = 0.30, P > 0.05) or g the cilia length (t = 0.30, P > 0.05, n = 4) in the caudal striatum. Scale bar = 10 μm. h–j ADCY3 immunostaining in the ventral striatum (nucleus accumbens). h Representative images of ADCY3 immunostaining in the ventral striatum showing that the selective removal of cilia from the rostral striatum does not affect i the number of ciliated neurons (t = 0.52, P > 0.05) or j the cilia length (t = 0.08, P > 0.05, n = 4) in the ventral striatum. Scale bar = 10 μm. k Effect of cilia removal on body weight to confirm normal gross growth (n = 8 control, 6 IFT88-KO). Unpaired t-test (t = 0.2463, P = 0.8096) revealed no significant difference in body weight. ns, not significant. Data are presented as means ± S.E.M. l Verification of well-being (n = 8 control, 6 IFT88-KO). Unpaired t-test (t = 0.2060, P = 0.8403) showed normal response to nociceptive stimulus. ns, not significant. Data are presented as means ± S.E.M
Primary cilia ablation in the striatum affects motor and sensorimotor-related behaviors. a Distance traveled in the last 60 min of the locomotor assay (n = 8 control, 6 IFT88-KO). Two-way ANOVA (F(11, 144) = 0.4143, P > 0.05) followed by Bonferroni’s post hoc test showed that IFT88-KO mice displayed similar locomotor activity to the control mice; ns, not significant. b Total locomotor activity in the locomotor assay. Unpaired t-test (t = 0.9608, P = 0.3556), ns, not significant. Data are presented as means ± S.E.M. c and d Motor skill learning on the accelerated rotarod. c Speed to fall in rotarod assay (n = 8). Two-way ANOVA (cilia removal factor: F(1,42) = 9.399, P = 0.0038, trial factor F(2,42) = 1.927, P > 0.05) followed by Bonferroni post hoc test: IFT88-KO vs control, **P < 0.001. Data are presented as means ± S.E.M; d Latency to fall in rotarod assay (n = 8). Two-way ANOVA (cilia removal factor: F(1,42) = 9.246, P = 0.0041, trial factor: F(2,42) = 1.93, P > 0.05) followed by Bonferroni post hoc test: IFT88-KO vs control, ***P < 0.001. Data are presented as means ± S.E.M. e Repetitive behavior in grooming behavior assays (n = 8 control, 6 IFT88-KO), unpaired t-test (t = 4.357, P = 0.0009), IFT88-KO vs control. Data are presented as means ± S.E.M. f and g Performance of mice in PPI assay. f Startle reactivity in prepulse inhibition assay (n = 8), unpaired t-test (t = 1.424, P = 0.1763), ns, not significant. g Prepulse inhibition in PPI assay (n = 8), two-way ANOVA (cilia removal factor: F(1,56) = 41.69, P < 0.0001, prepulse intensity factor: F(3,56) = 19.55, P < 0.0001), control vs IFT88-KO, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as means ± S.E.M
Primary cilia removal in the dorsal striatum does not affect anxiety, sociability, and depressive-like behaviors. a Time spent in central vs time in peripheral zones in open field assay (n = 8 control, 6 IFT88-KO), two-way ANOVA (cilia removal factor: F(1,24) = 0.00, P > 0.999, zone factor: F(1,24) = 538.1, P < 0.0001) followed by Bonferroni post hoc test. ****P < 0.0001. Data are presented as means ± S.E.M. b Total distance traveled in open field assay, unpaired t-test (t = 3.598, P = 0.0037), IFT88-KO vs control, **P < 0.01. Data are presented as means ± S.E.M. c Distance traveled in central vs time in peripheral zones in open field assay (n = 8 control, 6 IFT88-KO), two-way ANOVA (cilia removal factor: F(1,24) = 21.1, P = 0.0001, zone factor: F(1,24) = 68.31, P < 0.0001), followed by Bonferroni post hoc test, ****P < 0.0001. e Social interaction (n = 8), two-way ANOVA (cup factor: F(1,28) = 26.25, P < 0.0001, cilia removal factor: F(1,28) = 2.28, P = 0.1420); Empty cup vs stranger mouse, **P < 0.0001. Data are presented as means ± S.E.M. f Social interaction discrimination index, unpaired t-test (t = 0.2422, P = 0.8121), control vs IFT88-KO, ns, not significant. Data are presented as means ± S.E.M
Primary cilia in the striatum are required for spatial working memory but not other memories. a Spatial working memory alteration percentage in T-maze (n = 8 control, 6 IFT88-KO), unpaired t-test (t = 7.679, P < 0.0001), control vs IFT88-KO, ****P < 0.0001, b Decision latency in T-maze test (n = 8 control, 6 IFT88-KO), unpaired t-test (t = 5.295, P = 0.0002), control vs IFT88-KO, ***P < 0.001, c social novelty recognition (n = 8), two-way ANOVA (cilia removal factor: F(1,28) = 10.90, P = 0.0026, novel mouse factor: (F(1,28), P = 0.9612), followed by Bonferroni post hoc test: old mouse vs new mouse, **P < 001, ns, not significant. Data are presented as means ± S.E.M. d Discrimination index in social novelty recognition (n = 8), unpaired t-test (t = 3.604, P = 0.0029), control vs IFT88-KO, **P < 0.01. e Novel object recognition (n = 8), two-way ANOVA (object factor: F(1,28) = 21.24, P < 0.0001, cilia removal factor: F(1,28) = 0.02662, P = 0.8716): old object vs new object: *P < 0.05, **P < 0.01. Data are presented as means ± S.E.M. f Discrimination index in novel object recognition (n = 8), unpaired t-test (t = 0.90, P = 0.38), control vs IFT88-KO, ns, not significant. Data are presented as means ± S.E.M. g Novel location recognition (n = 8), two-way ANOVA (object factor: F(1,28) = 20.30, P < 0.0001, cilia removal factor: F(1,28) = 0.2.46, P = 0.8716): *P < 0.05, **P < 0.01. h Discrimination index in novel location recognition (n = 8), unpaired t-test (t = 0.46, P = 0.65), control vs IFT88-KO, ns, not significant. i Fear conditioning (n = 8), two-way ANOVA (P > 0.05) followed by Bonferroni post hoc test: control vs. IFT88-KO. ns, not significant. Data are presented as means ± S.E.M
Effects of cilia removal in the dorsal striatum on cFos expression in the striatum, its input and output structures. a Schematic view of neuronal circuits in mice brain. Amy, amygdala; CTX, cortex; GPL, lateral globus pallidus; GPm, medial globus pallidus; Hipp, hippocampus; Hyp, hypothalamus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; STN, subthalamic nucleus; STR, striatum; THA, thalamus; VTN, ventral tegmental area. Diagram was created with the BioRender.com webpage. b cFos immunostaining in four levels of the mouse brain. c Representative images of cFos immunostaining in the striatum and its input and output structures: striatum (STR), substantia nigra pars compacta (SNc), substantia nigra pars reticulata (SNr), lateral globus pallidus (GPl), medial globus pallidus (GPm), subthalamic nucleus (STN), prefrontal cortex (PFC), primary motor cortex (PMC), secondary motor cortex (SMC), primary somatosensory area (PSSA). Scale bar = 10 μm. d Quantification of the cFos-positive cells in the control and IFT88-KO mice. Two-way ANOVA, control vs IFT88-KO (F(1,88) = 94.28, P < 0.0001), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. Data are presented as means ± S.E.M; n = 3 sections of 5 mice per group
Almost all brain cells contain cilia, antennae-like microtubule-based organelles. Yet, the significance of cilia, once considered vestigial organelles, in the higher-order brain functions is unknown. Cilia act as a hub that senses and transduces environmental sensory stimuli to generate an appropriate cellular response. Similarly, the striatum, a brain structure enriched in cilia, functions as a hub that receives and integrates various types of environmental information to drive appropriate motor response. To understand cilia’s role in the striatum functions, we used loxP/Cre technology to ablate cilia from the dorsal striatum of male mice and monitored the behavioral consequences. Our results revealed an essential role for striatal cilia in the acquisition and brief storage of information, including learning new motor skills, but not in long-term consolidation of information or maintaining habitual/learned motor skills. A fundamental aspect of all disrupted functions was the “time perception/judgment deficit.” Furthermore, the observed behavioral deficits form a cluster pertaining to clinical manifestations overlapping across psychiatric disorders that involve the striatum functions and are known to exhibit timing deficits. Thus, striatal cilia may act as a calibrator of the timing functions of the basal ganglia-cortical circuit by maintaining proper timing perception. Our findings suggest that dysfunctional cilia may contribute to the pathophysiology of neuro-psychiatric disorders, as related to deficits in timing perception.
 
Accumulating clinical and epidemiological studies indicate that learning and memory impairment is more prevalent among people with diabetes mellitus (DM). PTP1B is a member of protein tyrosine phosphatase family and participates in a variety of pathophysiological effects including inflammatory, insulin signaling pathway, and learning and memory. This study was aimed to investigate the effects of CA, a specific inhibitor of PTP1B, on spatial learning and memory impairment in diabetic mice caused by high-fat diet and injection of streptozotocin. We found that the protein expressions of PTP1B increased in hippocampal CA1, CA3, and PFC regions of diabetic mice. Network pharmacology results showed that PTP1B might be one of the key targets between diabetes and cognitive dysfunction, and CA might alleviate DM-induced cognitive dysfunction. Animal experiments showed that CA ameliorated DM-induced spatial learning and memory impairment, and improved glucose and lipid metabolic disorders. Moreover, administration of CA alleviated hippocampal structure damage and enhanced the expressions of synaptic proteins, including PSD-95, SYN-1, and SYP in diabetic mice. Furthermore, CA treatment not only significantly down-regulated the expressions of PTP1B and NLRP3 inflammatory related proteins (NLRP3, ASC, Caspase-1, COX-2, IL-1β, and TNF-α), but also significantly up-regulated the expressions of insulin signaling pathway–related proteins (p-IRS1, p-PI3K, p-AKT, and p-GSK-3β) in diabetic mice. Taken together, these results suggested that PTP1B might be a targeted strategy to rescue learning and memory deficits in DM, possibly through inhibition of NLRP3 inflammasome and regulation of insulin signaling pathway.