Signatures of natural selection between life cycle stages separated by metamorphosis in European eel

Received: 16 December 2014, Accepted: 6 July 2015, Published: 13 August 2015[Background] Species showing complex life cycles provide excellent opportunities to study the genetic associations between life cycle stages, as selective pressures may differ before and after metamorphosis. The European eel presents a complex life cycle with two metamorphoses, a first metamorphosis from larvae into glass eels (juvenile stage) and a second metamorphosis into silver eels (adult stage). We tested the hypothesis that different genes and gene pathways will be under selection at different life stages when comparing the genetic associations between glass eels and silver eels.[Results] We used two sets of markers to test for selection: first, we genotyped individuals using a panel of 80 coding-gene single nucleotide polymorphisms (SNPs) developed in American eel; second, we investigated selection at the genome level using a total of 153,423 RAD-sequencing generated SNPs widely distributed across the genome. Using the RAD approach, outlier tests identified a total of 2413 (1.57 %) potentially selected SNPs. Functional annotation analysis identified signal transduction pathways as the most over-represented group of genes, including MAPK/Erk signalling, calcium signalling and GnRH (gonadotropin-releasing hormone) signalling. Many of the over-represented pathways were related to growth, while others could result from the different conditions that eels inhabit during their life cycle.[Conclusions] The observation of different genes and gene pathways under selection when comparing glass eels vs. silver eels supports the adaptive decoupling hypothesis for the benefits of metamorphosis. Partitioning the life cycle into discrete morphological phases may be overall beneficial since it allows the different life stages to respond independently to their unique selection pressures. This might translate into a more effective use of food and niche resources and/or performance of phase-specific tasks (e.g. feeding in the case of glass eels, migrating and reproducing in the case of silver eels).We acknowledge funding from the Danish Council for Independent Reasearch, Natural Sciences (grant 09-072120 to MMH).Peer reviewe

The Unfolded Protein Response: A Key Player in Zika Virus-Associated Congenital Microcephaly

Zika virus (ZIKV) is a mosquito-borne virus that belongs to the Flaviviridae family, together with dengue, yellow fever, and West Nile viruses. In the wake of its emergence in the French Polynesia and in the Americas, ZIKV has been shown to cause congenital microcephaly. It is the first arbovirus which has been proven to be teratogenic and sexually transmissible. Confronted with this major public health challenge, the scientific and medical communities teamed up to precisely characterize the clinical features of congenital ZIKV syndrome and its underlying pathophysiological mechanisms. This review focuses on the critical impact of the unfolded protein response (UPR) on ZIKV-associated congenital microcephaly. ZIKV infection of cortical neuron progenitors leads to high endoplasmic reticulum (ER) stress. This results in both the stalling of indirect neurogenesis, and UPR-dependent neuronal apoptotic death, and leads to cortical microcephaly. In line with these results, the administration of molecules inhibiting UPR prevents ZIKV-induced cortical microcephaly. The discovery of the link between ZIKV infection and UPR activation has a broader relevance, since this pathway plays a crucial role in many distinct cellular processes and its induction by ZIKV may account for several reported ZIKV-associated defects

Local delivery of interleukin 7 with an oncolytic adenovirus activates tumor-infiltrating lymphocytes and causes tumor regression

Cytokines have proven to be effective for cancer therapy, however whilst low-dose monotherapy with cytokines provides limited therapeutic benefit, high-dose treatment can lead to a number of adverse events. Interleukin 7 has shown promising results in clinical trials, but anti-cancer effect was limited, in part due to a low concentration of the cytokine within the tumor. We hypothesized that arming an oncolytic adenovirus with Interleukin 7, enabling high expression localized to the tumor microenvironment, would overcome systemic delivery issues and improve therapeutic efficacy. We evaluated the effects of Ad5/3-E2F-d24-hIL7 (TILT-517) on tumor growth, immune cell activation and cytokine profiles in the tumor microenvironment using three clinically relevant animal models and ex vivo tumor cultures. Our data showed that local treatment of tumor bearing animals with Ad5/3- E2F-d24-hIL7 significantly decreased cancer growth and increased frequency of tumor-infiltrating cells. Ad5/3-E2F-d24-hIL7 promoted notable upregulation of pro-inflammatory cytokines, and concomitant activation and migration of CD4+ and CD8 + T cells. Interleukin 7 expression within the tumor was positively correlated with increased number of cytotoxic CD4+ cells and IFNg-producing CD4+ and CD8+ cells. These findings offer an approach to overcome the current limitations of conventional IL7 therapy and could therefore be translated to the clinic.Peer reviewe

Importin-8 Modulates Division of Apical Progenitors, Dendritogenesis and Tangential Migration During Development of Mouse Cortex

The building of the brain is a multistep process that requires the coordinate expression of thousands of genes and an intense nucleocytoplasmic transport of RNA and proteins. This transport is mediated by karyopherins that comprise importins and exportins. Here, we investigated the role of the ß-importin, importin-8 (IPO8) during mouse cerebral corticogenesis as several of its cargoes have been shown to be essential during this process. First, we showed that Ipo8 mRNA is expressed in mouse brain at various embryonic ages with a clear signal in the sub-ventricular/ventricular zone (SVZ/VZ), the cerebral cortical plate (CP) and the ganglionic eminences. We found that acute knockdown of IPO8 in cortical progenitors reduced both their proliferation and cell cycle exit leading to the increase in apical progenitor pool without influencing the number of basal progenitors (BPs). Projection neurons ultimately reached their appropriate cerebral cortical layer, but their dendritogenesis was specifically affected, resulting in neurons with reduced dendrite complexity. IPO8 knockdown also slowed the migration of cortical interneurons. Together, our data demonstrate that IPO8 contribute to the coordination of several critical steps of cerebral cortex development. These results suggest that the impairment of IPO8 function might be associated with some diseases of neuronal migration defects

Bacurd2 is a novel interacting partner to Rnd2 which controls distinct phases of radial migration within the developing mammalian cerebral cortex

Within the developing mammalian cerebral cortex, newborn excitatory neurons engage in radial migra9on as they leave their birthplace in the germinal ventricular zone and reach their final loca9on before undergoing terminal differen9a9on. We have previously reported that the atypical RhoA GTPase Rnd2 promotes the radial migra9on of newborn cor9cal projec9on neurons within the embryonic cerebral cortex (REF), but its downstream signalling pathways are not well understood. In this study, we iden9fy Bacurd2 (a member of the BTB-domain containing adaptor for Cul3- mediated RhoA degrada9on) as a novel interac9ng partner to Rnd2 which promotes radial migra9on within the mouse cerebral cortex during embryonic and post-natal development. We find that Bacurd2 binds Rnd2 at its C-terminus, and this interac9on is cri9cal to its role in cell migra9on. To inves9gate how the interac9on between Bacurd2 and Rnd2 might be important for cell migra9on within the embryonic cortex, we engineered a Bacurd2:Rnd2 chimeric construct and discovered that the migra9on- defect of Rnd2shRNA-treated cells could be corrected by co-delivery of this construct. Our cellular analysis further reveals that Bacurd2-Rnd2 signalling is cri9cal for coordina9ng the mul9polar-to-bipolar transi9on of neurons within the intermediate zone, as well as their radial migra9on within the cor9cal plate. Therefore, our results iden9fy Bacurd2 as a cri9cal player during cerebral cor9cal development which guides the proper posi9oning of newborn neurons through its interac9on with Rnd2.Bacurd is an interacting partner to Rnd2 which controls radial migration within the developing cerebral corte

Bacurd2 is a novel player during cortical development, which influences the migration and morphological differentiation of cerebral cortical neurons

Members of the Bacurd (BTB-domain containing adaptor for Cul3-mediated RhoA degradation) proteins are implicated in neurological disorders such as Autism Spectrum Disorders, but their functions during brain development remain poorly understood. In this study, we describe a novel role for Bacurd2 during mouse cerebral cortical development, with forced expression and knockdown of Bacurd2 significantly disrupting the migration of cortical cells in embryonic mouse brains. In neuritogenic assays, overexpression of Bacurd2 in PC12 cells results in significant impairment of neuritogenesis. We further demonstrate the protein-protein interacting terminal domains of Bacurd2 to possess complementary functions for neuritogenesis, with Rnd2 and Cul3 as interacting partners to its carboxy and amino termini, respectively. Although suppression of Rnd2 expression by RNAi impairs cell migration in vivo[2,3], we were not able to rescue the migration defect of Rnd2-deficient cortical neurons with wild type Bacurd2 alone but only with chimeric Bacurd2 polypeptides that localise to the perinuclear region of Rnd2. Further analysis of the migration profile showed defective entry of Rnd2-deficient cells into the cortical plate when co-electroporated with chimeric Bacurd2 polypeptide that lack Cul3 binding ability. Our results highlight a novel mechanism for Cul3-Bacurd2-Rnd2 interaction that regulates morphology of neurons, as well as mediating radial migration of immature neurons during cerebral cortical development.Bacurd2 is a novel interacting partner to Rnd2 which controls radial migration within the developing mammalian cerebral corte

Stress-induced unfolded protein response contributes to Zika virus-associated microcephaly

Accumulating evidence support a causal link between Zika virus (ZIKV) infection during pregnancy and congenital microcephaly. However, the mechanism of ZIKV- associated microcephaly remains unclear. We combined analyses of ZIKV-infected human foetuses, cultured human neural stem cells and mouse embryos to understand how ZIKV induces microcephaly. We show here that ZIKV triggers ER stress and UPR in the cerebral cortex of infected postmortem human foetuses as well as in cultured human neural stem cells. After intracerebral and intraplacental inoculation of ZIKV in mouse embryos, we also show that it triggers endoplasmic reticulum stress in embryonic brains in vivo. This perturbs a physiological unfolded protein response within cortical progenitors that controls neurogenesis. Thus, ZIKV-infected progenitors generate fewer projection neurons that eventually settle in the cerebral cortex whereupon sustained ER stress leads to apoptotosis. Furthermore, we demonstrate that administration of pharmacological inhibitors of UPR counteracts these pathophysiological mechanisms, and prevents microcephaly in ZIKV-infected mouse embryos. Such defects are specific to ZIKV as they were not observed upon intraplacental injection of other related flaviviruses in mice.Stress-induced unfolded protein response contributes to Zika virus-associated microcephal

Identification and characterisation of novel genetic factors which promote neuronal differentiation during cerebral cortex development

The human cerebral cortex is highly adapted to process complex information. It plays a crucial role in the control of cognitive function, consciousness and intelligent behaviour [1-4]. These functions are dependent on the proper development of the cerebral cortex, which at its early stages involves the appropriate proliferation of progenitors and generation of postmitotic neurons [2, 5, 6]. Newborn neurons need to migrate to reach their appropriate position within the developing neocortex where they undergo terminal differentiation and form appropriate synaptic connections [7, 8]. During the course of neurodevelopment, excitatory projection neurons are generated from progenitors in the ventricular zone of the neocortex, and these migrate radially towards more superficial layers. Defects in neuronal migration lead to altered neuronal positioning and laminar patterning of the cortex, which can disrupt the assembly of functional cortical circuits [9-11]. The developmental processes of neuronal migration, dendritic arborisation and synaptic connectivity are controlled by the coordinated expressions of genes, and mediated through the activities of DNA binding transcription factors (TFs) [12-14]. However, the precise molecular mechanisms that control TF activity in neuronal development within the mammalian cerebral cortex remain poorly characterised. Recent studies have shown that the transcription activator Neurogenin2 (Ngn2) [15, 16] and transcriptional Repressor Protein 58 (Rp58) [17, 18] coordinate neuronal migration in the developing cortex by regulating the expression of downstream target genes, including Rnd2 [15-18]. Specifically, it was found that Ngn2 controls the migration of embryonic cortical neurons through activation of Rnd2 expression [15, 16], whereas Rp58 suppresses Rnd2 as neurons complete their radial migration [17, 18]. While these studies highlight the interplay between these transcriptional regulators for the control of cell migration, what remains less well understood is the signalling pathways of Rnd2 in neuronal development, and the interactions between gene regulatory pathways of Ngn2 and Rp58, both of which likely specify other aspects of neuronal development, such as the dendritic differentiation of cortical neurons. The focus of this thesis is to elucidate the molecular mechanisms that guide the radial migration and terminal differentiation of cerebral cortical neurons. In studies which address the protein signalling pathway for Rnd2 during neuronal development, I have identified Bacurd2, a member of the BTB-domain containing adaptor for Cul3-mediated RhoA degradation, as a novel interacting partner which promotes radial migration within the embryonic mouse cerebral cortex. I demonstrate that the interaction between Bacurd2 and Rnd2 is crucial for their combined roles in cell migration in vivo. My cellular analysis provides a basis for understanding the combined roles for Bacurd2 and Rnd2 in order to coordinate the multipolar-to-bipolar (MP-to-BP) transition of neurons as they migrate within the embryonic cortex. In further exploration of the biological functions for Bacurd2, and its related family member Bacurd1, I present evidence to suggest that disruptions to either of these genes impair the differentiation of neurons. Finally, I describe the generation of transgenic Rp58 mouse lines to study the development of post-migratory neurons within the cerebral cortex. Altogether, this thesis provides new insights into the development of cerebral cortical neurons and identifies Bacurds as new players in this process

Identification and characterisation of novel genetic factors which promote neuronal differentiation during cerebral cortex development

The human cerebral cortex is highly adapted to process complex information. It plays a crucial role in the control of cognitive function, consciousness and intelligent behaviour [1-4]. These functions are dependent on the proper development of the cerebral cortex, which at its early stages involves the appropriate proliferation of progenitors and generation of postmitotic neurons [2, 5, 6]. Newborn neurons need to migrate to reach their appropriate position within the developing neocortex where they undergo terminal differentiation and form appropriate synaptic connections [7, 8]. During the course of neurodevelopment, excitatory projection neurons are generated from progenitors in the ventricular zone of the neocortex, and these migrate radially towards more superficial layers. Defects in neuronal migration lead to altered neuronal positioning and laminar patterning of the cortex, which can disrupt the assembly of functional cortical circuits [9-11]. The developmental processes of neuronal migration, dendritic arborisation and synaptic connectivity are controlled by the coordinated expressions of genes, and mediated through the activities of DNA binding transcription factors (TFs) [12-14]. However, the precise molecular mechanisms that control TF activity in neuronal development within the mammalian cerebral cortex remain poorly characterised. Recent studies have shown that the transcription activator Neurogenin2 (Ngn2) [15, 16] and transcriptional Repressor Protein 58 (Rp58) [17, 18] coordinate neuronal migration in the developing cortex by regulating the expression of downstream target genes, including Rnd2 [15-18]. Specifically, it was found that Ngn2 controls the migration of embryonic cortical neurons through activation of Rnd2 expression [15, 16], whereas Rp58 suppresses Rnd2 as neurons complete their radial migration [17, 18]. While these studies highlight the interplay between these transcriptional regulators for the control of cell migration, what remains less well understood is the signalling pathways of Rnd2 in neuronal development, and the interactions between gene regulatory pathways of Ngn2 and Rp58, both of which likely specify other aspects of neuronal development, such as the dendritic differentiation of cortical neurons. The focus of this thesis is to elucidate the molecular mechanisms that guide the radial migration and terminal differentiation of cerebral cortical neurons. In studies which address the protein signalling pathway for Rnd2 during neuronal development, I have identified Bacurd2, a member of the BTB-domain containing adaptor for Cul3-mediated RhoA degradation, as a novel interacting partner which promotes radial migration within the embryonic mouse cerebral cortex. I demonstrate that the interaction between Bacurd2 and Rnd2 is crucial for their combined roles in cell migration in vivo. My cellular analysis provides a basis for understanding the combined roles for Bacurd2 and Rnd2 in order to coordinate the multipolar-to-bipolar (MP-to-BP) transition of neurons as they migrate within the embryonic cortex. In further exploration of the biological functions for Bacurd2, and its related family member Bacurd1, I present evidence to suggest that disruptions to either of these genes impair the differentiation of neurons. Finally, I describe the generation of transgenic Rp58 mouse lines to study the development of post-migratory neurons within the cerebral cortex. Altogether, this thesis provides new insights into the development of cerebral cortical neurons and identifies Bacurds as new players in this process

Ketamine selectively enhances AMPA neurotransmission onto a subgroup of identified serotoninergic neurons of the rat dorsal raphe

ABSTRACTAlthough the fast antidepressant effect of ketamine is now well established clinically, neither its mechanism(s) nor its main site(s) of action is clearly defined. Because enhanced serotoninergic (5-HT) transmission is an important part of the antidepressant effect of various drug classes, we asked whether ketamine and one of its metabolites (hydroxynorketamine [HNK]), both used in their racemic form, may modulate the excitatory drive onto these neurons.Using whole-cell recordings from pharmacologically identified 5-HT and non-5-HT neurons in juvenile rat dorsal raphe slices, we found that both ketamine and HNK (10 µM) increase excitatory AMPA neurotransmission onto a subset (50%) of 5-HT neurons, whereas other 5-HT cells were unaffected. Both compounds increased the amplitude as well as the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) mediated by AMPA receptors. The effect of ketamine was more robust than the one of HNK, since it significantly enhanced the charge transfer through AMPA channels, whereas HNK did not. The increase in the excitatory drive induced by ketamine was dependent on NMDA receptor blockade. In the presence of tetrodotoxin, the effect of ketamine was markedly reduced. Non-5-HT neurons, on the other hand, were unaffected by the drugs.We conclude that ketamine and HNK increase the excitatory drive onto a subset of 5-HT neurons by promoting glutamate release and possibly also through a postsynaptic action. The effect of ketamine is dependent on NMDA receptor modulation and appears to involve a network effect. These findings improve our understanding of the fast-acting antidepressant effect of ketamine