DCLK1 mediated cooperative acceleration of EMT by avian leukosis virus subgroup J and Marek's disease virus via the Wnt/ß-catenin pathway promotes tumor metastasis.

Co-infection with oncogenic retrovirus and herpesvirus significantly facilitates tumor metastasis in human and animals. Co-infection with avian leukosis virus subgroup J (ALV-J) and Marek's disease virus (MDV), which are typical oncogenic retrovirus and herpesvirus, respectively, leads to enhanced oncogenicity and accelerated tumor formation, resulting in increased mortality of affected chickens. Previously, we found that ALV-J and MDV cooperatively promoted tumor metastasis. However, the molecular mechanism remains elusive. Here, we found that doublecortin-like kinase 1 (DCLK1) mediated cooperative acceleration of epithelial-mesenchymal transition (EMT) by ALV-J and MDV promoted tumor metastasis. Mechanistically, DCLK1 induced EMT via activating Wnt/ß-catenin pathway by interacting with ß-catenin, thereby cooperatively promoting tumor metastasis. Initially, we screened and found that DCLK1 was a potential mediator for the cooperative activation of EMT by ALV-J and MDV, and enhanced cell proliferation, migration, and invasion. Subsequently, we revealed that DCLK1 physically interacted with ß-catenin to promote the formation of the ß-catenin-TCF4 complex, inducing transcription of the Wnt target gene, c-Myc, promoting EMT by increasing the expression of N-cadherin, Vimentin, and Snail, and decreasing the expression of E-cadherin. Taken together, we discovered that jointly activated DCLK1 by ALV-J and MDV accelerated cell proliferation, migration and invasion, and ultimately activated EMT, paving the way for tumor metastasis. This study elucidated the molecular mechanism underlying cooperative metastasis induced by co-infection with retrovirus and herpesvirus. IMPORTANCE: Tumor metastasis, a complex phenomenon in which tumor cells spread to new organs, is one of the greatest challenges in cancer research and is the leading cause of cancer-induced death. Numerous studies have shown that oncoviruses and their encoded proteins significantly affect metastasis, especially the EMT process. ALV-J and MDV are classic tumorigenic retrovirus and herpesvirus, respectively. We found that ALV-J and MDV synergistically promoted EMT. Further, we identified the tumor stem cell marker DCLK1 in ALV-J and MDV co-infected cells. DCLK1 directly interacted with ß-catenin, promoting the formation of the ß-catenin-TCF4 complex. This interaction activated the Wnt/ß-catenin pathway, thereby inducing EMT and paving the way for synergistic tumor metastasis. Exploring the molecular mechanisms by which ALV-J and MDV cooperate during EMT will contribute to our understanding of tumor progression and metastasis. This study provides new insights into the cooperative induced tumor metastasis by retroviruses and herpesviruses.

Generation of a Dcx-CreERT2 knock-in mouse for genetic manipulation of newborn neurons.

A wide variety of CreERT2 driver lines are available for genetic manipulation of adult-born neurons in the mouse brain. These tools have been instrumental in studying fate potential, migration, circuit integration, and morphology of the stem cells supporting lifelong neurogenesis. Despite a wealth of tools, genetic manipulation of adult-born neurons for circuit and behavioral studies has been limited by poor specificity of many driver lines targeting early progenitor cells and by the inaccessibility of lines selective for later stages of neuronal maturation. We sought to address these limitations by creating a new CreERT2 driver line targeted to the endogenous mouse doublecortin locus as a marker of fate-specified neuroblasts and immature neurons. Our new model places a T2A-CreERT2 cassette immediately downstream of the Dcx coding sequence on the X chromosome, allowing expression of both Dcx and CreERT2 proteins in the endogenous spatiotemporal pattern for this gene. We demonstrate that the new mouse line drives expression of a Cre-dependent reporter throughout the brain in neonatal mice and in known neurogenic niches of adult animals. The line has been deposited with the Jackson Laboratory and should provide an accessible tool for studies targeting fate-restricted neuronal precursors.

Altered hippocampal doublecortin expression in Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative disorder characterized by motor and non-motor symptoms. Motor symptoms include bradykinesia, resting tremors, muscular rigidity, and postural instability, while non-motor symptoms include cognitive impairments, mood disturbances, sleep disturbances, autonomic dysfunction, and sensory abnormalities. Some of these symptoms may be influenced by the proper hippocampus functioning, including adult neurogenesis. Doublecortin (DCX) is a microtubule-associated protein that plays a pivotal role in the development and differentiation of migrating neurons. This study utilized postmortem human brain tissue of PD and age-matched control individuals to investigate DCX expression in the context of adult hippocampal neurogenesis. Our findings demonstrate a significant reduction in the number of DCX-expressing cells within the subgranular zone (SGZ), as well as a decrease in the nuclear area of these DCX-positive cells in postmortem brain tissue obtained from PD cases, suggesting an impairment in the adult hippocampal neurogenesis. Additionally, we found that the nuclear area of DCX-positive cells correlates with pH levels. In summary, we provide evidence supporting that the process of hippocampal adult neurogenesis is likely to be compromised in PD patients before cognitive dysfunction, shedding light on potential mechanisms contributing to the neuropsychiatric symptoms observed in affected individuals. Understanding these mechanisms may offer novel insights into the pathophysiology of PD and possible therapeutic avenues.

Estrogens dynamically regulate neurogenesis in the dentate gyrus of adult female rats.

Estrone and estradiol differentially modulate neuroplasticity and cognition. How they influence the maturation of new neurons in the adult hippocampus, however, is not known. The present study assessed the effects of estrone and estradiol on the maturation timeline of neurogenesis in the dentate gyrus (DG) of ovariectomized (a model of surgical menopause) young adult Sprague-Dawley rats using daily subcutaneous injections of 17ß-estradiol, estrone or vehicle. Rats were injected with a DNA synthesis marker, 5-bromo-2-deoxyuridine (BrdU), and were perfused 1, 2, or 3 weeks after BrdU injection and daily hormone treatment. Brains were sectioned and processed for various markers including: sex-determining region Y-box 2 (Sox2), glial fibrillary acidic protein (GFAP), antigen kiel 67 (Ki67), doublecortin (DCX), and neuronal nuclei (NeuN). Immunofluorescent labeling or co-labelling of BrdU with Sox2 (progenitor cells), Sox2/GFAP (neural progenitor cells), Ki67 (cell proliferation), DCX (immature neurons), NeuN (mature neurons) was used to examine the trajectory and maturation of adult-born neurons over time. Estrogens had early (1 week of exposure) effects on different stages of neurogenesis (neural progenitor cells, cell proliferation and early maturation of new cells into neurons) but these effects were less pronounced after prolonged treatment. Estradiol enhanced, whereas estrone reduced cell proliferation after 1 week but not after longer exposure to either estrogen. Both estrogens increased the density of immature neurons (BrdU/DCX-ir) after 1 week of exposure compared to vehicle treatment but this increased density was not sustained over longer durations of treatments to estrogens, suggesting that the enhancing effects of estrogens on neurogenesis were short-lived. Longer duration post-ovariectomy, without treatments with either of the estrogens, was associated with reduced neural progenitor cells in the DG. These results demonstrate that estrogens modulate several aspects of adult hippocampal neurogenesis differently in the short term, but may lose their ability to influence neurogenesis after long-term exposure. These findings have potential implications for treatments involving estrogens after surgical menopause.

Adrenergic microenvironment driven by cancer-associated Schwann cells contributes to chemoresistance in patients with lung cancer.

Doublecortin (DCX)-positive neural progenitor-like cells are purported components of the cancer microenvironment. The number of DCX-positive cells in tissues reportedly correlates with cancer progression; however, little is known about the mechanism by which these cells affect cancer progression. Here we demonstrated that DCX-positive cells, which are found in all major histological subtypes of lung cancer, are cancer-associated Schwann cells (CAS) and contribute to the chemoresistance of lung cancer cells by establishing an adrenergic microenvironment. Mechanistically, the activation of the Hippo transducer YAP/TAZ was involved in the acquisition of new traits of CAS and DCX positivity. We further revealed that CAS express catecholamine-synthesizing enzymes and synthesize adrenaline, which potentiates the chemoresistance of lung cancer cells through the activation of YAP/TAZ. Our findings shed light on CAS, which drive the formation of an adrenergic microenvironment by the reciprocal regulation of YAP/TAZ in lung cancer tissues.

Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography.

Neurons extend axons to form the complex circuitry of the mature brain. This depends on the coordinated response and continuous remodelling of the microtubule and F-actin networks in the axonal growth cone. Growth cone architecture remains poorly understood at nanoscales. We therefore investigated mouse hippocampal neuron growth cones using cryo-electron tomography to directly visualise their three-dimensional subcellular architecture with molecular detail. Our data showed that the hexagonal arrays of actin bundles that form filopodia penetrate and terminate deep within the growth cone interior. We directly observed the modulation of these and other growth cone actin bundles by alteration of individual F-actin helical structures. Microtubules with blunt, slightly flared or gently curved ends predominated in the growth cone, frequently contained lumenal particles and exhibited lattice defects. Investigation of the effect of absence of doublecortin, a neurodevelopmental cytoskeleton regulator, on growth cone cytoskeleton showed no major anomalies in overall growth cone organisation or in F-actin subpopulations. However, our data suggested that microtubules sustained more structural defects, highlighting the importance of microtubule integrity during growth cone migration.

Microglia depletion and repopulation do not alter the effects of cranial irradiation on hippocampal neurogenesis.

Cranial radiotherapy can cause lifelong cognitive complications in childhood brain tumor survivors, and reduced hippocampal neurogenesis is hypothesized to contribute to this. Following irradiation (IR), microglia clear dead neural progenitors and give rise to a neuroinflammatory microenvironment, which promotes a switch in surviving progenitors from neuronal to glial differentiation. Recently, depletion and repopulation of microglia were shown to promote neurogenesis and ameliorate cognitive deficits in various brain injury models. In this study, we utilized the Cx3cr1CreERt2-YFP/+Rosa26DTA/+ transgenic mouse model to deplete microglia in the juvenile mouse brain before subjecting them to whole-brain IR and investigated the short- and long-term effects on hippocampal neurogenesis. Within the initial 24 h after IR, the absence of microglia led to an accumulation of dead cells in the subgranular zone, and 50-fold higher levels of the chemokine C-C motif ligand 2 (CCL2) in sham brains and 7-fold higher levels after IR. The absence of microglia, and the subsequent repopulation within 10 days, did neither affect the loss of proliferating or doublecortin-positive cells, nor the reduced growth of the granule cell layer. Our results argue against a role for a pro-inflammatory microenvironment in the dysregulation of hippocampal neurogenesis and suggest that the observed reduction of neurogenesis was solely due to IR.

Discovery of a doublecortin-like kinase 1 inhibitor to prevent inflammatory responses in acute lung injury.

Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated protein kinase involved in neurogenesis and human cancer. Recent studies have revealed a novel functional role for DCLK1 in inflammatory signaling, thus positioning it as a novel target kinase for respiratory inflammatory disease treatment. In this study, we designed and synthesized a series of NVP-TAE684-based derivatives as novel anti-inflammatory agents targeting DCLK1. Bio-layer interferometry binding screening and kinase assays of the NVP-TAE684 derivatives led to the discovery of an effective DCLK1 inhibitor (a24), with an IC50 of 179.7 nM. Compound a24 effectively inhibited lipopolysaccharide (LPS)-induced inflammation in macrophages with higher potency than the lead compound. Mechanistically, compound a24 inhibited LPS-induced inflammation by inhibiting DCLK1-mediated IKKß phosphorylation. Furthermore, compound a24 showed in vivo anti-inflammatory activity in an LPS-challenged acute lung injury model. These findings suggest that compound a24 may serve as a novel candidate for the development of DCLK1 inhibitors and a potential therapeutic agent for the treatment of inflammatory diseases.

Proteomic investigation of neural stem cell to oligodendrocyte precursor cell differentiation reveals phosphorylation-dependent Dclk1 processing.

Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify doublecortin-like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. All data are available via ProteomeXchange with identifier PXD040652.

Design and synthesis of doublecortin-like kinase 1 inhibitors and their bioactivity evaluation.

Doublecortin-like kinase 1 (DCLK) is a microtubule-associated serine/threonine kinase that is upregulated in a wide range of cancers and is believed to be related to tumour growth and development. Upregulated DCLK1 has been used to identify patients at high risk of cancer progression and tumours with chemotherapy-resistance. Moreover, DCLK1 has been identified as a cancer stem cell (CSC) biomarker in various cancers, which has received considerable attention recently. Herein, a series of DCLK1 inhibitors were prepared based on the previously reported XMD8-92 structure. Among all the synthesised compounds, D1, D2, D6, D7, D8, D12, D14, and D15 showed higher DCLK1 inhibitory activities (IC50 40-74 nM) than XMD8-92 (IC50 161 nM). Compounds D1 and D2 were selective DCLK1 inhibitors as they showed a rather weak inhibitory effect on LRRK2. The antiproliferative activities of these compounds were also preliminarily evaluated. The structure-activity relationship revealed by our compounds provides useful guidance for the further development of DCLK1 inhibitors.

Phenotypic Variability in Novel Doublecortin Gene Variants Associated with Subcortical Band Heterotopia.

Doublecortin, encoded by the DCX gene, plays a crucial role in the neuronal migration process during brain development. Pathogenic variants of the DCX gene are the major causes of the "lissencephaly (LIS) spectrum", which comprehends a milder phenotype like Subcortical Band Heterotopia (SBH) in heterozygous female subjects. We performed targeted sequencing in three unrelated female cases with SBH. We identified three DCX-related variants: a novel missense (c.601A>G: p.Lys201Glu), a novel nonsense (c.210C>G: p.Tyr70*), and a previously identified nonsense (c.907C>T: p.Arg303*) variant. The novel c.601A>G: p.Lys201Glu variant shows a mother-daughter transmission pattern across four generations. The proband exhibits focal epilepsy and achieved seizure freedom with a combination of oxcarbazepine and levetiracetam. All other affected members have no history of epileptic seizures. Brain MRIs of the affected members shows predominant fronto-central SBH with mixed pachygyria on the overlying cortex. The two nonsense variants were identified in two unrelated probands with SBH, severe drug-resistant epilepsy and intellectual disability. These novel DCX variants further expand the genotypic-phenotypic correlations of lissencephaly spectrum disorders. Our documented phenotypic descriptions of three unrelated families provide valuable insights and stimulate further discussions on DCX-SBH cases.

Morphological Signatures of Neurogenesis and Neuronal Migration in Hypothalamic Vasopressinergic Magnocellular Nuclei of the Adult Rat.

The arginine vasopressin (AVP)-magnocellular neurosecretory system (AVPMNS) in the hypothalamus plays a critical role in homeostatic regulation as well as in allostatic motivational behaviors. However, it remains unclear whether adult neurogenesis exists in the AVPMNS. By using immunoreaction against AVP, neurophysin II, glial fibrillar acidic protein (GFAP), cell division marker (Ki67), migrating neuroblast markers (doublecortin, DCX), microglial marker (Ionized calcium binding adaptor molecule 1, Iba1), and 5'-bromo-2'-deoxyuridine (BrdU), we report morphological evidence that low-rate neurogenesis and migration occur in adult AVPMNS in the rat hypothalamus. Tangential AVP/GFAP migration routes and AVP/DCX neuronal chains as well as ascending AVP axonal scaffolds were observed. Chronic water deprivation significantly increased the BrdU+ nuclei within both the supraaoptic (SON) and paraventricular (PVN) nuclei. These findings raise new questions about AVPMNS's potential hormonal role for brain physiological adaptation across the lifespan, with possible involvement in coping with homeostatic adversities.

Constitutive Neurogenesis and Neuronal Plasticity in the Adult Cerebellum and Brainstem of Rainbow Trout, Oncorhynchus mykiss.

The central nervous system of Pacific salmon retains signs of embryonic structure throughout life and a large number of neuroepithelial neural stem cells (NSCs) in the proliferative areas of the brain, in particular. However, the adult nervous system and neurogenesis studies on rainbow trout, Oncorhynchus mykiss, are limited. Here, we studied the localization of glutamine synthetase (GS), vimentin (Vim), and nestin (Nes), as well as the neurons formed in the postembryonic period, labeled with doublecortin (DC), under conditions of homeostatic growth in adult cerebellum and brainstem of Oncorhynchus mykiss using immunohistochemical methods and Western Immunoblotting. We observed that the distribution of vimentin (Vim), nestin (Nes), and glutamine synthetase (GS), which are found in the aNSPCs of both embryonic types (neuroepithelial cells) and in the adult type (radial glia) in the cerebellum and the brainstem of trout, has certain features. Populations of the adult neural stem/progenitor cells (aNSPCs) expressing GS, Vim, and Nes have different morphologies, localizations, and patterns of cluster formation in the trout cerebellum and brainstem, which indicates the morphological and, obviously, functional heterogeneity of these cells. Immunolabeling of PCNA revealed areas in the cerebellum and brainstem of rainbow trout containing proliferating cells which coincide with areas expressing Vim, Nes, and GS. Double immunolabeling revealed the PCNA/GS PCNA/Vim coexpression patterns in the neuroepithelial-type cells in the PVZ of the brainstem. PCNA/GS coexpression in the RG was detected in the submarginal zone of the brainstem. The results of immunohistochemical study of the DC distribution in the cerebellum and brainstem of trout have showed a high level of expression of this marker in various cell populations. This may indicate: (i) high production of the adult-born neurons in the cerebellum and brainstem of adult trout, (ii) high plasticity of neurons in the cerebellum and brainstem of trout. We assume that the source of new cells in the trout brain, along with PVZ and SMZ, containing proliferating cells, may be local neurogenic niches containing the PCNA-positive and silent (PCNA-negative), but expressing NSC markers, cells. The identification of cells expressing DC, Vim, and Nes in the IX-X cranial nerve nuclei of trout was carried out.

Involvement of brain-derived neurotrophic factor methylation in the prefrontal cortex and hippocampus induced by chronic unpredictable mild stress in male mice.

Early life stress alters brain-derived neurotrophic factor (BDNF) promoter IV methylation and BDNF expression, which is closely related to the pathophysiological process of depression. However, the role of abnormal methylation of BDNF induced by stress during adolescence due to depression has not yet been clarified. In this study, adolescent mice were exposed to chronic unpredictable mild stress (CUMS). Depression-like behaviors, BDNF promoter IV methylation, expression of DNA methyltransferases (DNMTs), demethylation machinery enzymes, BDNF protein levels, and neuronal development in the prefrontal cortex (PFC) and hippocampus (HIP) were assessed in adolescent and adult mice. The DNMT inhibitor, 5-Aza-2-deoxycytidine (5-AzaD), was used as an intervention. Stress in adolescence induces behavioral dysfunction, elevated methylation levels of BDNF promoter IV, changes in the expression of DNMT, and demethylation machinery enzymes in adolescent and adult mice. Additionally, the stress in adolescence induced lower levels of BDNF and abnormal hippocampal doublecortin (DCX) expression in adolescent and adult mice. However, DNMT inhibitor treatment in adolescent-stressed mice relieved the abnormal behaviors, normalized the methylation level of BDNF promoter IV, BDNF protein expression, expression of DNMTs, and demethylation machinery enzymes, and improved the neuronal development of adult mice. These results suggest that stress in adolescence induces short- and long-term hypermethylation of BDNF promoter IV, which is regulated by DNMTs, and leads to the development of depression.

A complex relation between levels of adult hippocampal neurogenesis and expression of the immature neuron marker doublecortin.

We investigated the mechanisms underlying the effects of the antidepressant fluoxetine on behavior and adult hippocampal neurogenesis (AHN). After confirming our earlier report that the signaling molecule ß-arrestin-2 (ß-Arr2) is required for the antidepressant-like effects of fluoxetine, we found that the effects of fluoxetine on proliferation of neural progenitors and survival of adult-born granule cells are absent in the ß-Arr2 knockout (KO) mice. To our surprise, fluoxetine induced a dramatic upregulation of the number of doublecortin (DCX)-expressing cells in the ß-Arr2 KO mice, indicating that this marker can be increased even though AHN is not. We discovered two other conditions where a complex relationship occurs between the number of DCX-expressing cells compared to levels of AHN: a chronic antidepressant model where DCX is upregulated and an inflammation model where DCX is downregulated. We concluded that assessing the number of DCX-expressing cells alone to quantify levels of AHN can be complex and that caution should be applied when label retention techniques are unavailable.

Interaction between Avian Leukosis Virus Subgroup J Surface Protein and Doublecortin-Like Kinase 1 Accelerates Cell Proliferation and Epithelial-Mesenchymal Transition.

Avian leukosis virus subgroup J (ALV-J) induces myelocytomas, which can metastasize to multiple organs in diseased chickens. Although metastasis is the primary cause of death in such cases, the mechanism for it remains unclear. Here, we found that interaction between ALV-J surface protein (SU) and doublecortin-like kinase 1 (DCLK1) promotes epithelial-mesenchymal transition (EMT) and cell proliferation. We found that ALV-J can activate EMT in infected cells. Subsequently, proteomics analysis revealed that DCLK1, a well-established putative tumor stem cell marker, which is highly expressed in ALV-J-infected DF-1 cells and chickens, might be a potential factor mediating EMT. Furthermore, using immunofluorescence and immunoprecipitation, we verified that SU interacts with DCLK1. Functional studies suggested that overexpression of DCLK1 increased viral replication and promoted cell proliferation by accelerating the progression of cells from the G0/G1 phase to the S phase of cell cycle, whereas RNA interference of DCLK1 reduced viral replication and arrested cell proliferation by retarding cell cycle progression from the late G1 phase into the S phase in ALV-J-infected cells. Moreover, we demonstrate that the increased accumulation of DCLK1 promotes EMT by increasing the expression of N-cadherin, vimentin, MMP2, and transcription factor Snail1 and decreasing the expression of epithelial marker E-cadherin. These results suggest that ALV-J SU interacts with DCLK1, and accelerates cell proliferation, leading to increased viral replication and ultimately activating EMT, which paves the way for tumor metastasis. IMPORTANCE Tumor metastasis is a major challenge in cancer research, because of its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents. It is estimated that >90% of mortality from cancer is attributable to metastases. We found that ALV-J can activate EMT, which plays a critical role in cancer metastasis. Subsequently, we identified a tumor stem cell marker, DCLK1, in ALV-J infected cells, which interacts with surface protein (SU) of ALV-J to promote virus replication, activate EMT, and accelerate cell proliferation enabling ALV-J to obtain metastatic ability. Understanding the process of participation of ALV-J in EMT and the route of metastasis will help elucidate the mechanism of virus-induced tumor metastasis and help identify promising molecular targets and key obstacles for ALV-J control and clinical technology development.

Biallelic known and novel DCDC2 variants in cholestatic liver disease: Phenotype-genotype observations in four children.

Neonatal sclerosing cholangitis (NSC) is associated with progressing biliary fibrosis that often requires liver transplantation in childhood. Several recent studies have identified variants in DCDC2, encoding doublecortin domain-containing protein 2 (DCDC2), expressed in primary cilia, that accompany syndromic disease and NSC. We report four patients with hepatobiliary disease associated with two novel homozygous or compound heterozygous variants in DCDC2. Three patients with protein-truncating variants in DCDC2, expressing no DCDC2, presented with the originally described severe hepatic phenotype in infancy. One patient with a novel homozygous DCDC2 missense variant shows a markedly milder phenotype only manifest in childhood and with retained DCDC2 expression. Concomitant nephronophthisis is present in three patients and learning disability in two. This report widens the phenotypic spectrum of DCDC2-associated hepatobiliary disease. Testing for DCDC2 expression and DCDC2 variants should be included in the evaluation of cholangiopathy of unknown aetiology in childhood as well as in infancy.

Hippocampal neurogenesis and Arc expression are enhanced in high-fat fed prepubertal female pigs by a diet including omega-3 fatty acids and Bifidobacterium breve CECT8242.

PURPOSE: Obesity during childhood has become a pandemic disease, mainly caused by a diet rich in sugars and fatty acids. Among other negative effects, these diets can induce cognitive impairment and reduce neuroplasticity. It is well known that omega-3 and probiotics have a beneficial impact on health and cognition, and we have hypothesized that a diet enriched with Bifidobacterium breve and omega-3 could potentiate neuroplasticity in prepubertal pigs on a high-fat diet. METHODS: Young female piglets were fed during 10 weeks with: standard diet (T1), high-fat (HF) diet (T2), HF diet including B. breve CECT8242 (T3) and HF diet including the probiotic and omega-3 fatty acids (T4). Using hippocampal sections, we analyzed by immunocytochemistry the levels of doublecortin (DCX) to study neurogenesis, and activity-regulated cytoskeleton-associated protein (Arc) as a synaptic plasticity related protein. RESULTS: No effect of T2 or T3 was observed, whereas T4 increased both DCX+ cells and Arc expression. Therefore, a diet enriched with supplements of B. breve and omega-3 increases neurogenesis and synaptic plasticity in prepubertal females on a HF diet from nine weeks of age to sexual maturity. Furthermore, the analysis of serum cholesterol and HDL indicate that neurogenesis was related to lipidic demand in piglets fed with control or HF diets, but the neurogenic effect induced by the T4 diet was exerted by mechanisms independent of this lipidic demand. CONCLUSION: Our results show that the T4 dietary treatment is effective in potentiating neural plasticity in the dorsal hippocampus of prepubertal females on a HF diet.

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.

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.