Doublecortin-like kinase 3 (DCLK3) is associated with bad clinical outcome of patients with gastric cancer and regulates the ferroptosis and mitochondria function in vitro and in vivo.

BACKGROUND: Doublecortin-like kinase 3 (DCLK3), a member of tubulin superfamily, has been proved to be closely associated with the pathogenesis of numerous human tumors. However, the expression pattern and regulatory mechanisms of DCLK3 in gastric cancer (GC) remain unknown. MATERIALS AND METHODS: DCLK3 expression in GC cells was assessed by RT-qPCR and western blotting. The correlation between DCLK3 levels and the overall survival of GC patients was assessed via TCGA, ACLBI, and Kaplan-Meier plotter databases. Additionally, key proteins (TCF4) involved in the regulation of DCLK3 on GC progression were screened by ACLBI database. Cell proliferation, ferroptotic cell death, and oxidative stress markers were measured by EdU staining, immunofluorescence, ELISA, and western blotting assays. RESULTS: DCLK3 was upregulated in GC, and high DCLK3 expression was significantly associated with poor survival of GC patients. Here, DCLK3 knockdown reduced GC cell proliferation, induced ferroptotic cell death, and exacerbated oxidative stress level. Logistic regression analysis showed that TCF4 was an independent prognostic indicator of GC. Mechanistically, DCLK3 promoted TCF4 expression and subsequently upregulated the expression of TCF4 downstream target genes (c-Myc and Cyclin D1). Furthermore, DCLK3 overexpression enhanced GC cell proliferation, but mitigating ferroptotic cell death and oxidative stress. The regulatory mechanism may involve the upregulation of TCF4, c-Myc, and cyclin D1. CONCLUSIONS: Our research suggests that DCLK3 modulates the levels of iron and reactive oxygen and may involve regulation of TCF4 pathway, thereby promoting the GC cell growth, indicating that DCLK3 may use as a prognostic marker and therapeutic target for GC patients.

Therapeutic hypothermia modulates the neurogenic response of the newborn piglet subventricular zone after hypoxia-ischemia.

BACKGROUND: Neuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy. To investigate the neurogenic response to hypoxia-ischemia (HI) followed by normothermia (38.5 °C) or three different hypothermic temperatures (35, 33.5, or 30 °C) in the subventricular zone (SVZ) of the neonatal piglet. METHODS: Following transient cerebral HI and resuscitation, 28 newborn piglets were randomized to: normothermia or whole-body cooling to 35 °C, 33.5 °C, or 30 °C during 2-26 h (all n = 7). At 48 h, piglets were euthanized and SVZ obtained to evaluate its cellularity, pattern of cell death, radial glia length, doublecortin (DCX, neuroblasts) expression, and Ki67 (cell proliferation) and Ki67/Sox2 (neural stem/progenitor dividing) cell counts. RESULTS: Normothermic piglets showed lower total (Ki67+) and neural stem/progenitor dividing (Ki67+Sox2+) cell counts when compared to hypothermic groups. Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and DCX immunohistochemistry. Cooling to 30 °C, however, revealed decreased cellularity in the lateral SVZ and shorter radial glia processes when compared with 33.5 °C. CONCLUSIONS: In a neonatal piglet model, hypothermia to 33.5 °C modulates the neurogenic response of the SVZ after HI, highlighting the potential beneficial effect of hypothermia to 33.5 °C on endogenous neurogenesis and the detrimental effect of overcooling beyond this threshold. IMPACT: Neuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy. Hypothermia may modulate neurogenesis in the subventricular zone (SVZ) of the neonatal hypoxic-ischemic piglet. Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and doublecortin immunohistochemistry; cooling to 30 °C, however, revealed decreased cellularity and shorter radial glia processes. In a neonatal piglet model, therapeutic hypothermia (33.5 °C) modulates the neurogenic response of the SVZ after hypoxia-ischemia, highlighting also the detrimental effect of overcooling beyond this threshold.

Melatonin Prevents Depression but Not Anxiety-like Behavior Produced by the Chemotherapeutic Agent Temozolomide: Implication of Doublecortin Cells and Hilar Oligodendrocytes.

Melatonin is a hormone synthesized by the pineal gland with neuroprotective and neurodevelopmental effects. Also, melatonin acts as an antidepressant by modulating the generation of new neurons in the dentate gyrus of the hippocampus. The positive effects of melatonin on behavior and neural development may suggest it is used for reverting stress but also for the alterations produced by chemotherapeutic drugs influencing behavior and brain plasticity. In this sense, temozolomide, an alkylating/anti-proliferating agent used in treating brain cancer, is associated with decreased cognitive functions and depression. We hypothesized that melatonin might prevent the effects of temozolomide on depression- and anxiety-like behavior by modulating some aspects of the neurogenic process in adult Balb/C mice. Mice were treated with temozolomide (25 mg/kg) for three days of two weeks, followed by melatonin (8 mg/kg) for fourteen days. Temozolomide produced short- and long-term decrements in cell proliferation (Ki67-positive cells: 54.89% and 53.38%, respectively) and intermediate stages of the neurogenic process (doublecortin-positive cells: 68.23% and 50.08%, respectively). However, melatonin prevented the long-term effects of temozolomide with the increased number of doublecortin-positive cells (47.21%) and the immunoreactivity of 2' 3'-Cyclic-nucleotide-3 phosphodiesterase (CNPase: 82.66%), an enzyme expressed by mature oligodendrocytes, in the hilar portion of the dentate gyrus. The effects of melatonin in the temozolomide group occurred with decreased immobility in the forced swim test (45.55%) but not anxiety-like behavior. Thus, our results suggest that melatonin prevents the harmful effects of temozolomide by modulating doublecortin cells, hilar oligodendrocytes, and depression-like behavior tested in the forced swim test. Our study could point out melatonin's beneficial effects for counteracting temozolomide's side effects.

Zn2+ decoration of microtubules arrests axonal transport and displaces tau, doublecortin, and MAP2C.

Intracellular Zn2+ concentrations increase via depolarization-mediated influx or intracellular release, but the immediate effects of Zn2+ signals on neuron function are not fully understood. By simultaneous recording of cytosolic Zn2+ and organelle motility, we find that elevated Zn2+ (IC50 ≈ 5-10 nM) reduces both lysosomal and mitochondrial motility in primary rat hippocampal neurons and HeLa cells. Using live-cell confocal microscopy and in vitro single-molecule TIRF imaging, we reveal that Zn2+ inhibits activity of motor proteins (kinesin and dynein) without disrupting their microtubule binding. Instead, Zn2+ directly binds to microtubules and selectively promotes detachment of tau, DCX, and MAP2C, but not MAP1B, MAP4, MAP7, MAP9, or p150glued. Bioinformatic predictions and structural modeling show that the Zn2+ binding sites on microtubules partially overlap with the microtubule binding sites of tau, DCX, dynein, and kinesin. Our results reveal that intraneuronal Zn2+ regulates axonal transport and microtubule-based processes by interacting with microtubules.

Repetitive transcranial magnetic stimulation and fluoxetine reverse depressive-like behavior but with differential effects on Olig2-positive cells in chronically stressed mice.

Depression is a mood disorder coursing with several behavioral, cellular, and neurochemical alterations. The negative impact of chronic stress may precipitate this neuropsychiatric disorder. Interestingly, downregulation of oligodendrocyte-related genes, abnormal myelin structure, and reduced numbers and density of oligodendrocytes in the limbic system have been identified in patients diagnosed with depression, but also in rodents exposed to chronic mild stress (CMS). Several reports have emphasized the importance of pharmacological or stimulation-related strategies in influencing oligodendrocytes in the hippocampal neurogenic niche. Repetitive transcranial magnetic stimulation (rTMS) has gained attention as an intervention to revert depression. Here, we hypothesized that 5 Hz (Hz) of rTMS or Fluoxetine (Flx) would revert depressive-like behaviors by influencing oligodendrocytes and revert neurogenic alterations caused by CMS in female Swiss Webster mice. Our results showed that 5 Hz rTMS or Flx revert depressive-like behavior. Only rTMS influenced oligodendrocytes by increasing the number of Olig2-positive cells in the hilus of the dentate gyrus and the prefrontal cortex. However, both strategies exerted effects on some events of the hippocampal neurogenic processes, such as cell proliferation (Ki67-positive cells), survival (CldU-positive cells), and intermediate stages (doublecortin-positive cells) along the dorsal-ventral axis of this region. Interestingly, the combination of rTMS-Flx exerted antidepressant-like effects, but the increased number of Olig2-positive cells observed in mice treated only with rTMS was canceled. However, rTMS-Flx exerted a synergistic effect by increasing the number of Ki67-positive cells. It also increased the number of CldU- and doublecortin-positive cells in the dentate gyrus. Our results demonstrate that 5 Hz rTMS has beneficial effects, as it reverted depressive-like behavior by increasing the number of Olig2-positive cells and reverting the decrement in hippocampal neurogenesis in CMS-exposed mice. Nevertheless, the effects of rTMS on other glial cells require further investigation.

Sevoflurane Induces a Cyclophilin D-Dependent Decrease of Neural Progenitor Cells Migration.

Clinical studies have suggested that repeated exposure to anesthesia and surgery at a young age may increase the risk of cognitive impairment. Our previous research has shown that sevoflurane can affect neurogenesis and cognitive function in young animals by altering cyclophilin D (CypD) levels and mitochondrial function. Neural progenitor cells (NPCs) migration is associated with cognitive function in developing brains. However, it is unclear whether sevoflurane can regulate NPCs migration via changes in CypD. To address this question, we treated NPCs harvested from wild-type (WT) and CypD knockout (KO) mice and young WT and CypD KO mice with sevoflurane. We used immunofluorescence staining, wound healing assay, transwell assay, mass spectrometry, and Western blot to assess the effects of sevoflurane on CypD, reactive oxygen species (ROS), doublecortin levels, and NPCs migration. We showed that sevoflurane increased levels of CypD and ROS, decreased levels of doublecortin, and reduced migration of NPCs harvested from WT mice in vitro and in WT young mice. KO of CypD attenuated these effects, suggesting that a sevoflurane-induced decrease in NPCs migration is dependent on CypD. Our findings have established a system for future studies aimed at exploring the impacts of sevoflurane anesthesia on the impairment of NPCs migration.

Doublecortin-Expressing Neurons in Human Cerebral Cortex Layer II and Amygdala from Infancy to 100 Years Old.

A cohort of morphologically heterogenous doublecortin immunoreactive (DCX +) "immature neurons" has been identified in the cerebral cortex largely around layer II and the amygdala largely in the paralaminar nucleus (PLN) among various mammals. To gain a wide spatiotemporal view on these neurons in humans, we examined layer II and amygdalar DCX + neurons in the brains of infants to 100-year-old individuals. Layer II DCX + neurons occurred throughout the cerebrum in the infants/toddlers, mainly in the temporal lobe in the adolescents and adults, and only in the temporal cortex surrounding the amygdala in the elderly. Amygdalar DCX + neurons occurred in all age groups, localized primarily to the PLN, and reduced in number with age. The small-sized DCX + neurons were unipolar or bipolar, and formed migratory chains extending tangentially, obliquely, and inwardly in layers I-III in the cortex, and from the PLN to other nuclei in the amygdala. Morphologically mature-looking neurons had a relatively larger soma and weaker DCX reactivity. In contrast to the above, DCX + neurons in the hippocampal dentate gyrus were only detected in the infant cases in parallelly processed cerebral sections. The present study reveals a broader regional distribution of the cortical layer II DCX + neurons than previously documented in human cerebrum, especially during childhood and adolescence, while both layer II and amygdalar DCX + neurons persist in the temporal lobe lifelong. Layer II and amygdalar DCX + neurons may serve as an essential immature neuronal system to support functional network plasticity in human cerebrum in an age/region-dependent manner.

Doublecortin and Glypican-2 concentrations in the cerebrospinal fluid from infants are developmentally downregulated.

OBJECTIVE: Doublecortin (DCX) and glypican-2 (GPC2) are neurodevelopmental proteins involved in the differentiation of neural stem/progenitor cells (NSPCs) to neurons, and are developmentally downregulated in neurons after birth. In this study, we investigated whether the concentrations of DCX and GPC2 in the cerebrospinal fluid (CSF) from human pediatric patients reflect this developmental process or are associated with cerebral damage or inflammatory markers. METHODS: CSF was collected from pediatric patients requiring neurosurgical treatment. The concentrations of DCX, GPC2, neuron-specific enolase (NSE), S100 calcium-binding protein B (S100B), and cytokines (IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IFN-γ, and TNF-⍺) were measured using immunoassays. RESULTS: From March 2013 until October 2018, 63 CSF samples were collected from 38 pediatric patients (20 females; 17 patients with repeated measurements); the median term born-adjusted age was 3.27 years [Q1: 0.31, Q3: 7.72]. The median concentration of DCX was 329 pg/ml [Q1: 192.5, Q3: 1179.6] and that of GPC2 was 26 pg/ml [Q1: 13.25, Q3: 149.25]. DCX and GPC2 concentrations independently significantly associated with age, and their concentration declined with advancing age, reaching undetectable levels at 0.3 years for DCX, and plateauing at 1.5 years for GPC2. Both DCX and GPC2 associated with hydrocephalus, NSE, IL-1ß, IL-2, IL-8, IL-13. No relationship was found between sex, acute infection, S100B, IL-4, IL-6, IL-10, IFN-γ, TNF-α and DCX or GPC2, respectively. CONCLUSIONS: Concentrations of DCX and GPC2 in the CSF from pediatric patients are developmentally downregulated, with the highest concentrations measured at the earliest adjusted age, and reflect a neurodevelopmental stage rather than a particular disease state.

Repeated administration of acrylamide for 28 days suppresses adult neurogenesis of the olfactory bulb in young-adult rats.

Acrylamide (AA) is a neurotoxicant that inhibits synaptic function in distal axons. We previously found that AA decreased neural cell lineages during late-stage differentiation of adult hippocampal neurogenesis and downregulated genes related to neurotrophic factor, neuronal migration, neurite outgrowth, and synapse formation in the hippocampal dentate gyrus in rats. To investigate whether olfactory bulb (OB)-subventricular zone (SVZ) neurogenesis is similarly affected by AA exposure, AA was administered to 7-week-old male rats via oral gavage at doses of 0, 5, 10, and 20 mg/kg for 28 days. Immunohistochemical analysis revealed that AA decreased the numbers of doublecortin-positive (+) cells and polysialic acid-neural cell adhesion molecule+ cells in the OB. On the other hand, the numbers of doublecortin+ cells and polysialic acid-neural cell adhesion molecule+ cells in the SVZ did not change with AA exposure, suggesting that AA impaired neuroblasts migrating in the rostral migratory stream and OB. Gene expression analysis in the OB revealed that AA downregulated Bdnf and Ncam2, which are related to neuronal differentiation and migration. These results suggest that AA decreased neuroblasts in the OB by suppressing neuronal migration. Thus, AA decreased neuronal cell lineages during late-stage differentiation of adult neurogenesis in the OB-SVZ, similar to the effect on adult hippocampal neurogenesis.

Consistency and Variation in Doublecortin and Ki67 Antigen Detection in the Brain Tissue of Different Mammals, including Humans.

Recently, a population of "immature" neurons generated prenatally, retaining immaturity for long periods and finally integrating in adult circuits has been described in the cerebral cortex. Moreover, comparative studies revealed differences in occurrence/rate of different forms of neurogenic plasticity across mammals, the "immature" neurons prevailing in gyrencephalic species. To extend experimentation from laboratory mice to large-brained mammals, including humans, it is important to detect cell markers of neurogenic plasticity in brain tissues obtained from different procedures (e.g., post-mortem/intraoperative specimens vs. intracardiac perfusion). This variability overlaps with species-specific differences in antigen distribution or antibody species specificity, making it difficult for proper comparison. In this work, we detect the presence of doublecortin and Ki67 antigen, markers for neuronal immaturity and cell division, in six mammals characterized by widely different brain size. We tested seven commercial antibodies in four selected brain regions known to host immature neurons (paleocortex, neocortex) and newly born neurons (hippocampus, subventricular zone). In selected human brains, we confirmed the specificity of DCX antibody by performing co-staining with fluorescent probe for DCX mRNA. Our results indicate that, in spite of various types of fixations, most differences were due to the use of different antibodies and the existence of real interspecies variation.

Cu,Zn-Superoxide Dismutase has Minimal Effects Against Cuprizone-Induced Demyelination, Microglial Activation, and Neurogenesis Defects in the C57BL/6 Mouse Hippocampus.

Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.

Beneficial effects of the phytoestrogen genistein on hippocampal impairments of spontaneously hypertensive rats (SHR).

Hippocampal neuropathology is a recognized feature of the spontaneously hypertensive rat (SHR). The hippocampal alterations associate with cognitive impairment. We have shown that hippocampal abnormalities are reversed by 17ß-estradiol, a steroid binding to intracellular receptors (estrogen receptor α and ß subtypes) or the membrane-located G-protein coupled estradiol receptor. Genistein (GEN) is a neuroprotective phytoestrogen which binds to estrogen receptor ß and G-protein coupled estradiol receptor. Here, we investigated whether GEN neuroprotection extends to SHR. For this purpose, we treated 5-month-old SHR for 2 weeks with 10 mg kg-1 daily s.c injections of GEN. We analyzed the expression of doublecortin+ neuronal progenitors, glial fibrillary acidic protein+ astrocytes and ionized calcium-binding adapter molecule 1+ microglia in the CA1 region and dentate gyrus of the hippocampus using immunocytochemistry, whereas a quantitative real-time polymerase chain reaction was used to measure the expression of pro- and anti-inflammatory factors tumor necrosis factor α, cyclooxygenase-2 and transforming growth factor ß. We also evaluated hippocampal dependent memory using the novel object recognition test. The results showed a decreased number of doublecortin+ neural progenitors in the dentate gyrus of SHR that was reversed with GEN. The number of glial fibrillary acidic protein+ astrocytes in the dentate gyrus and CA1 was increased in SHR but significantly decreased by GEN treatment. Additionally, GEN shifted microglial morphology from the predominantly activated phenotype present in SHR, to the more surveillance phenotype found in normotensive rats. Furthermore, treatment with GEN decreased the mRNA of the pro-inflammatory factors tumor necrosis factor α and cyclooxygenase-2 and increased the mRNA of the anti-inflammatory factor transforming growth factor ß. Discrimination index in the novel object recognition test was decreased in SHR and treatment with GEN increased this parameter. Our results indicate important neuroprotective effects of GEN at the neurochemical and behavioral level in SHR. Our data open an interesting possibility for proposing this phytoestrogen as an alternative therapy in hypertensive encephalopathy.

Doublecortin immunolabeling in canine gliomas with distinct degrees of tumor infiltration.

Increased doublecortin (DCX) immunolabeling at the tumor margins has been associated with tumor infiltration in human glioma and canine anaplastic meningioma. No association between DCX immunolabeling and glioma infiltration has been reported in dogs, to our knowledge. Here we compare the DCX immunolabeling in 14 diffusely infiltrating gliomas (gliomatosis cerebri) and 14 nodular gliomas with distinct degrees of tumor infiltration. Cytoplasmic DCX immunolabeling was classified according to intensity (weak, moderate, strong), distribution (1 = <30% immunolabeling, 2 = 30-70% immunolabeling, 3 = >70% immunolabeling), and location within the neoplasm (random or at tumor margins). Immunolabeling was detected in 6 of 14 (43%) diffusely infiltrating gliomas and 8 of 14 (57%) nodular gliomas. Diffusely infiltrating gliomas had moderate and random immunolabeling, with distribution scores of 1 (4 cases) or 2 (2 cases). Nodular gliomas had strong (6 cases) or moderate (2 cases) immunolabeling, with distribution scores of 1 (3 cases), 2 (3 cases), and 3 (2 cases), and random (6 cases) and/or marginal (3 cases) immunolabeling. Increased DCX immunolabeling within neoplastic cells palisading around necrosis occurred in 4 nodular gliomas. DCX immunolabeling was not increased at the margins of diffusely infiltrating gliomas, indicating that DCX should not be used as an immunomarker for glioma infiltration in dogs.

Adiponectin Promotes Neurogenesis After Transient Cerebral Ischemia Through STAT3 Mediated BDNF Upregulation in Astrocytes.

Newborn neurons from the subventricular zone (SVZ) are essential to functional recovery following ischemic stroke. However, the number of newly generated neurons after stroke is far from enough to support a potent recovery. Adiponectin could increase neurogenesis in the dentate gyrus of hippocampus in neurodegenerative diseases. However, the effect of adiponectin on the neurogenesis from SVZ and the functional recovery after ischemic stroke was unknown, and the underlying mechanism was not specified either. The middle cerebral artery occlusion model of mice was adopted and adiponectin was administrated once a day from day 3 to 7 of reperfusion. The levels of BDNF and p-STAT3 were detected by western blotting on day 7 of reperfusion. The virus-encoded BDNF shRNA with GFAP promoter and a STAT3 inhibitor Stattic were used, respectively. Neurogenesis was evidenced by the expression of doublecortin and 5-bromo-2'-deoxyuridine (BrdU) labelling and brain atrophy was revealed by Nissl staining on day 28 of reperfusion. Neurological functional recovery was assessed by the adhesive removal test and the forepaw grip strength. We found that adiponectin increased both the doublecortin-positive cells and NeuN/BrdU double-positive cells around the injured area on day 28 of reperfusion, along with the improved long-term neurological recovery. Mechanistically, adiponectin increased the protein levels of p-STAT3 and BDNF in astrocytes on day 7 of reperfusion, while silencing BDNF diminished the adiponectin-induced neurogenesis and functional recovery. Moreover, inhibition of STAT3 not only prevented the increase of BDNF but also the improved neurogenesis and functional recovery after stroke. In conclusion, adiponectin enhances neurogenesis and functional recovery after ischemic stroke via STAT3/BDNF pathway in astrocytes.

Repeated administration of acrylamide for 28 days reduces late-stage progenitor cells and immature granule cells accompanying impaired neurite outgrowth in the adult hippocampal neurogenesis in young-adult rats.

Acrylamide (AA) is a neurotoxicant that causes synaptic impairment in distal axons. We previously found that developmental exposure to AA decreased proliferation of late-stage neural progenitor cells (NPCs) in the hippocampal neurogenesis of the dentate gyrus (DG) in rats. To investigate whether hippocampal neurogenesis is similarly affected by AA exposure in a general toxicity study, AA was administered to 7-week-old male rats via oral gavage at dosages of 0, 5, 10, and 20 mg/kg for 28 days. In the subgranular zone (SGZ) and granule cell layer, AA decreased the densities of doublecortin-positive (+) cells and TOAD-64/Ulip/CRMP protein 4b+ cells per SGZ length. In addition, AA decreased the neurite length of doublecortin+ cells and downregulated genes related to neurite outgrowth (Ncam2 and Nrep) and neurotrophic factor (Bdnf and Ntrk2) in the DG. These results suggest that AA exposure for 28 days decreases type-3 NPCs and immature granule cells in neurogenesis of granule cell lineages involving the impairment of neurite outgrowth in young-adult rats. In the DG hilus, AA increased the density of cholinergic receptor nicotinic beta 2 subunit+ cells. AA also downregulated Reln related to the control of neuronal migration by interneurons in the DG. Furthermore, AA decreased the density of glial fibrillary acidic protein (GFAP)+ astrocytes in the DG hilus and downregulated Gfap and the genes of oligodendrocyte progenitor cells (Cspg4 and Pdgfra). Thus, AA decreased granule cell lineage subpopulations in the late-stage differentiation of hippocampal neurogenesis after young-adult stage exposure, exhibiting a pattern similar to the developmental exposure.

Muscone with Attenuation of Neuroinflammation and Oxidative Stress Exerts Antidepressant-Like Effect in Mouse Model of Chronic Restraint Stress.

Major depressive disorder (MDD) is a common mental disorder with high morbidity. Stress negatively affects for MDD development, whereby transport of stress-induced inflammatory mediators to the central nervous system (CNS) is associated with the etiology of mood disorders. Muscone is a pharmacologically active ingredient isolated from musk, with anti-inflammatory and neuroprotective effects. We hypothesized that muscone may ameliorate depression-like behavior by regulating inflammatory responses. To test this hypothesis, we used the chronic restraint stress (CRS) depression model, and CRS mice were treated with muscone (10 mg/kg, i.g., respectively) for 14 days. The effects of the drug on depressive-like behaviors were evaluated via the open field test (OFT), novelty-suppressed feeding test (NSFT), tail suspension test (TST), and forced swimming test (FST). Quantitative reverse transcription-PCR (qRT-PCR) was utilized to assess levels of proinflammatory cytokines (IL-6, TNF-α, COX2, and IL-1) and the anti-inflammatory cytokines (IL-4 and IL-10). We also determined levels of oxidative stress factors (malondialdehyde, superoxide dismutase, and glutathione peroxidase), as well as doublecortin (DCX) expression by immunofluorescence. The results showed that depression-like behavior and inflammatory levels were improved after muscone treatment. Muscone also significantly improved neurogenesis in the CRS mouse hippocampus and decreased oxidative stress in both the central and peripheral nervous systems. In conclusion, this work is the first to demonstrate that muscone has an antidepressant effect using a CRS model. Oxidative stress, neurogenesis, and inflammatory pathways are key factors affected by the drug and may represent new therapeutic targets to treat MDD, in this impact. These results may represent a new therapeutic target for MDD.

Investigation of Naoluoxintong on the neural stem cells by facilitating proliferation and differentiation in vitro and on protecting neurons by up-regulating the expression of nestin in MCAO rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The classic traditional Chinese compound Naoluoxintong (NLXT) has been proven an effective remedy for ischemic stroke (IS). The protective effect of NLXT on neural stem cells (NSCs), however, remains unclear. AIM OF THE STUDY: To investigate the protective effect of NLXT on NSCs in rats with middle cerebral artery occlusion (MCAO) and the effect of Nestin expression in vivo. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: the sham-operated group, the MCAO model group and the NLXT group. The MCAO model in rats was established by modified Longa wire embolization method. The sham-operated group, the model group and the NLXT groups were divided into three subgroups according to the sampling time points of 1 d, 3 d and 7 d after successful model-making. Immunofluorescence staining, including bromodeoxyuridine (BrdU)/glial fibrillary acidic protein (GFAP), ß-tubulinIII/GFAP, BrdU/doublecortin (DCX) and BrdU/neuronal nuclei (NeuN), was used to detect the proliferation and survival of NSCs in the hippocampal after drug administration. Protein expression of Nestin, DCX, GFAP and NeuN in the hippocampal was detected by Western blot (WB). RESULTS: Immunofluorescence experiment of Nestin labeled: on the first day, a few Nestin-positive cells were found in the hippocampal DG area. Afterwards, the number of Nestin-labeled positive cells in the model group increased, while the number of cells in the sham group did not fluctuate significantly. The number of positive cells in each administration group increased more than that in the model and normal group. ß-tubulin III/GFAP double-labeled: a small amount of double labeled cells was expressed in the normal group, and the number subsequently fluctuated little. In the model group, ß-tubulin III/GFAP positive cells increased initially after acute ischemia, and gradually decreased afterwards. In the NLXT-treated group, ß-Tubulin III positive cells were significantly increased on day 1, 3 and 7, while GFAP positive cells had little change. BrdU/DCX double-labeled: initially, a small number of BrdU/DCX-labeled positive cells were observed in the normal group and the model group, but there was no increasing trend over time. The positive cells in the NLXT group increased over time, and those in the seven-day group were significantly higher than those in the one-day and three-day groups. BrdU/NEUN double-labeled: in the normal group, BrdU/NEUN positive cells were enriched and distributed regularly. The number of positive cells in the model group was small and decreased gradually with time, and the decrease was most obvious on the third day. The number of positive cells in the NLXT group was significantly higher than that in the model group, and the number of positive cells in the seven-day group was significantly higher than that in the one-day and three-day groups. WB results reflected those three proteins, Nestin, NeuN and DCX, showed an increase in expression, except GFAP, which showed a decreasing trend. CONCLUSIONS: Preliminarily, NLXT can promote the migration and differentiation of NSCs. It may have a protective effect on the brain by promoting repair of brain tissue damage through upregulation of Nestin after IS.

Expression of Transcription Factor ZBTB20 in the Adult Primate Neurogenic Niche under Physiological Conditions or after Ischemia.

The Zbtb20 gene encodes for a transcription factor that plays an important role in mammalian cortical development. Recently, its expression was reported in the adult mouse subventricular zone (SVZ), a major neurogenic niche containing neural stem cells throughout life. Here, we analyzed its expression in the adult primate anterior SVZ (SVZa) and rostral migratory stream (RMS) using macaque monkeys (Macaca fuscata). We report that the majority of Ki67+ cells, 71.4% in the SVZa and 85.7% in the RMS, co-label for ZBTB20. Nearly all neuroblasts, identified by their Doublecortin expression, were positive for ZBTB20 in both regions. Nearly all GFAP+ neural stem cells/astrocytes were also positive for ZBTB20. Analysis of images derived from a public database of gene expression in control/ischemic monkey SVZa, showed evidence for ZBTB20 upregulation in postischemic monkey SVZa. Furthermore, the co-localization of ZBTB20 with Doublecortin and Ki67 was increased in the postischemic SVZa. Our results suggest that ZBTB20 expression is evolutionarily conserved in the mammalian neurogenic niche and is reactive to ischemia. This opens the possibility for further functional studies on the role of this transcription factor in neurogenesis in primates.

Poor body condition is associated with lower hippocampal plasticity and higher gut methanogen abundance in adult laying hens from two housing systems.

It is still unclear which commercial housing system provides the best quality of life for laying hens. In addition, there are large individual differences in stress levels within a system. Hippocampal neurogenesis or plasticity may provide an integrated biomarker of the stressors experienced by an individual. We selected 12 adult hens each with good and poor body condition (based on body size, degree of feather cover and redness of the comb) from a multi-tier free range system containing H&N strain hens, and from an enriched cage system containing Hy-Line hens (n = 48 total). Immature neurons expressing doublecortin (DCX) were quantified in the hippocampus, contents of the caecal microbiome were sequenced, and expression of inflammatory cytokines was measured in the spleen. DCX+ cell densities did not differ between the housing systems. In both systems, poor condition hens had lower DCX+ cell densities, exhibited elevated splenic expression of interleukin-6 (IL6) mRNA, and had a higher relative caecal abundance of methanogenic archea Methanomethylophilaceae. The findings suggest poor body condition is an indicator that individual hens have experienced a comparatively greater degree of cumulative chronic stress, and that a survey of the proportion of hens with poor body conditions might be one way to evaluate the impact of housing systems on hen welfare.

Cerebral dopamine neurotrophic factor increases proliferation, Migration and differentiation of subventricular zone neuroblasts in photothrombotic stroke model of mouse.

BACKGROUND: Cerebral ischemic stroke can induce the proliferation of subventricular zone (SVZ) neural stem cells (NSCs) in the adult brain. However, this reparative process is restricted because of NSCs' death shortly after injury or disability of them to reach the infarct boundary. In the present study, we investigated the ability of cerebral dopamine neurotrophic factor (CDNF) on the attraction of SVZ-resident NSCs toward the lesioned area and neurological recovery in a photothrombotic (PT) stroke model of mice METHODS: The mice were assigned to three groups stroke, stroke+phosphate buffered saline (PBS), and stroke+CDNF. Migration of SVZ NSCs were evaluated by BrdU/doublecortin (DCX) double immunofluorescence method on days 7 and 14 and their differentiation were evaluated by BrdU/ Neuronal Nuclei (NeuN) double immunofluorescence method 28 days after intra-SVZ CDNF injection. Serial coronal sections were stained with cresyl violet to detect the infarct volume and a modified neurological severity score (mNSS) was performed to assess the neurological performance RESULTS: Injection of CDNF increased the proliferation of SVZ NSCs and the number of DCX-expressing neuroblasts migrated from the SVZ toward the ischemic site. It also enhanced the differentiation of migrated neuroblasts into the mature neurons in the lesioned site. Along with this, the infarct volume was significantly decreased and the neurological performance was improved as compared to other groups CONCLUSION: These results demonstrate that CDNF is capable of enhancing the proliferation of NSCs residing in the SVZ and their migration toward the ischemia region and finally, differentiation of them in stroke mice, concomitantly decreased infarct volume and improved neurological abilities were revealed.