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The time-sensitive GABA shift from excitatory to inhibitory is critical in early neural circuits development and depends upon developmentally regulated expression of cation-chloride cotransporters NKCC1 and KCC2. NKCC1, encoded by the SLC12A2 gene, regulates neuronal Cl− homeostasis by chloride import working opposite KCC2. The high NKCC1/KCC2 expression ratio decreases in early neural development contributing to GABA shift. Human SLC12A2 loss-of-function mutations were recently associated with a multisystem disorder affecting neural development. However, the multisystem phenotype of rodent Nkcc1 knockout models makes neurodevelopment challenging to study. Brain-Derived Neurotrophic Factor (BDNF)-NTRK2/TrkB signalling controls KCC2 expression during neural development, but its impact on NKCC1 is still controversial. Here, we discuss recent evidence supporting BDNF-TrkB signalling controlling Nkcc1 expression and the GABA shift during hippocampal circuit formation. Namely, specific deletion of Ntrk2/Trkb from immature mouse hippocampal dentate granule cells (DGCs) affects their integration and maturation in the hippocampal circuitry and reduces Nkcc1 expression in their target region, the CA3 principal cells, leading to premature GABA shift, ultimately influencing the establishment of functional hippocampal circuitry and animal behaviour in adulthood. Thus, immature DGCs emerge as a potential therapeutic target as GABAergic transmission is vital for specific neural progenitors generating dentate neurogenesis in early development and the mature brain.

Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor β (NGFβ), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFβ or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFβ was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRC4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFβ-TRKA signaling in pathogen-specific host immunity.

Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signalling, plasticity and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signalling. The tyrosine kinase neurotrophin receptors (Trk) and the low-affinity p75 neurotrophin receptor (p75NTR) are enriched in neuronal lipid rafts together with the intermediates of downstream signalling pathways, suggesting a possible role of rafts in neurotrophin signalling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

The BDNF receptor, TrkB, is critical to limbic epileptogenesis, but the responsible downstream signaling pathways are unknown. We hypothesized that TrkB-dependent activation of phospholipase Cgamma1 (PLCgamma1) signaling is the key pathway and tested this in trkB(PLC/PLC) mice carrying a mutation (Y816F) that uncouples TrkB from PLCgamma1. Biochemical measures revealed activation of both TrkB and PLCgamma1 in hippocampi in the pilocarpine and kindling models in wild-type mice. PLCgamma1 activation was decreased in hippocampi isolated from trkB(PLC/PLC) compared with control mice. Epileptogenesis assessed by development of kindling was inhibited in trkB(PLC/PLC) compared with control mice. Long-term potentiation of the mossy fiber-CA3 pyramid synapse was impaired in slices of trkB(PLC/PLC) mice. We conclude that TrkB-dependent activation of PLCgamma1 signaling is an important molecular mechanism of limbic epileptogenesis. Elucidating signaling pathways activated by a cell membrane receptor in animal models of CNS disorders promises to reveal novel targets for specific and effective therapeutic intervention.

The striatum, consisting of enkephalin-positive (Penk+) and substance P-positive subpopulations of medium spiny neurons (MSNs), integrates and drives information and function of the basal ganglia circuits, which is crucial for maintaining normal movement. Preferential degeneration of the Penk+ MSNs has been observed at early stage of Huntington disease (HD). Recently, selective deletion of TrkB, high affinity receptor of brain derived neurotrophic factor (BDNF), specifically in the Penk+ MSNs (TrkbPENK-KO) led to an age-dependent spontaneous hyperlocomotion, but not to neuronal loss, indicating that TrkB is essential to maintain normal locomotor control. However, the underlying molecular mechanism still remains unknown. Dissecting the molecular changes induced in the Penk+ MSNs in the context of absence of TrkB is imperative to understand mechanism of normal locomotor maintenance. We, therefore, have performed transcriptomics analysis by next generation sequencing (RNA-seq) on the purified Penk+ MSNs with and without ablation of TrkB at two ages. Subsequent bioinformatics analysis has revealed early energy metabolism and mitochondrial dysfunction specifically to the Penk+ MSNs, leading to the severe energy deficiency at later stage. We have then validated hits from the RNA-seq data by optimising comprehensive validation approaches. Interestingly, the bioinformatics analysis led to the discovery of dopamine dysregulation in mutants at early age that could contribute to an age-dependent hyperlocomotor phenotype. Amongst the hits we validated, an enzyme involved in the glutathione metabolism, was identified as a key gene with unique expression pattern driving transcriptomic changes of the Penk+ MSNs. Furthermore, by creating a Cre-dependent shRNA lentiviral vector, we selectively knocked down specific gene expression in the Penk+ MSNs in vivo at presymptomatic age. Excitingly, this significantly rescued the hyperlocomotor phenotype of the TrkbPENK-KO mice. These findings add knowledge to the molecular mechanisms underlying normal locomotor maintenance, and they also highlight related pathway as a potential target for the development of novel treatments to movement disorders.

Canonically, adult neurogenesis occurs, in mammals, in two distinct regions of the brain—the subgranular zone of the dentate gyrus and the ventricular-subventricular zone (V-SVZ) of the lateral ventricle. The V-SVZ germinal niche is comprised, in part, by ependymal cells and the quiescent neural stem cells (NSCs) that lie near these cells. During neurogenesis, the quiescent NSCs change their transcriptional state and alter their cellular machinery to become activated NSCs. Together, quiescent and activated NSCs are also known as B-cells. B-cells generate transit amplifying cells, which then generate neuroblasts. Initially, it was believed that the function of V-SVZ neurogenesis was to populate the olfactory bulb with new neurons. However, recent work suggests that under disease and injury states it can lead to the formation of new striatal neurons. Studying B-cells is important because although they were identified two decades ago, many important questions regarding their development, specification, and function remain unanswered. The fate of B-cells is specified at the time of their formation during embryonic development. This work suggested that there existed many different subpopulations of B-cells—each fated to generate different types of neurons in the adult mouse brain. However, the mechanism by which the B-cells are apportioned to the different subgroups still remains a mystery. In this thesis, we demonstrate that a BAC-Penk-CRE transgenic line (which causes CRE expression in cells with an active pre-pro-Enkephalin promoter), generated in our lab and crossed to an appropriate reporter strain, labels a novel subgroup of B-cells, as well as previously identified subgroups of B-cells and neuroblasts. Using single-cell RNA sequencing, and other validation techniques, we show that this novel subgroup expresses the 5-Hydroxytryptamine Receptor 2C (5-HT2C receptor) and the prolactin receptor—which have been functionally correlated with B-cells, but B-cells expressing these receptors have never been captured and sequenced before. We also show that this novel subgroup expresses the KiR7.1 channel and the folate receptor. These findings are novel and exciting and will allow for the further study of the heterogeneity of B-cells and to investigate the many unanswered questions. Finally, not only have we been able to validate the RNAseq data, but we also show that in a partial progressive model of Parkinson’s disease, cells expressing the BAC-Penk-CRE reporter increase their proliferation and can be found in the striatal parenchyma. Thereby demonstrating the functional importance of this group of V-SVZ stem cells.

The central nervous system (CNS) has a crucial role in the maintenance of energy homeostasis by orchestrating a plethora of signals from peripheral organs about the state of energy stores and the current energy intake needed to match energy expenditure. These signals converge into the hypothalamic regions and its complex local circuitry. CNS-derived cholecystokinin (CCK) is acting at central level to modulate energy balance by regulating the neuronal activity of hypothalamic neuronal populations that regulate food intake, energy storage and consumption. Moreover, our recent published work identifies CCK neurons as key integrators of the neuroendocrine negative feedback of glucocorticoids to the PVN. Glucose sensing neurons of the Ventromedial Hypothalamus (VMH) are integrating energy signals and are essential for mounting a counter-regulatory response and glucose homeostasis. VMH is also important in energy expenditure by regulating body weight and thermogenesis. CCK neurons are present in high density in the VMH.The source of endogenous CCK that acts on distinct neuronal components has not been elucidated. The research so far does not address the purpose of CCK neurons in the hypothalamus and their potential role in the network dynamics regarding energy homeostasis. In this study, we untangle the role of CCK neurons in the VMH nucleus by employing stereotactic intracranial delivery of adeno-associated viruses that result in cell-type specific chemogenetic inhibition or ablation of these neurons. Acute silencing of their neurotransmission with the cre-dependent AAV expression of the chemogenetic tool of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) increases their daily food intake due to increased meal numbers and eating frequency without meal size or meal duration being affected. CCK ablation by a newly generated double-recombinase-mediated Diphtheria Toxin Receptor (DTR) mouse line or AAV-DTA-mediated ablation resulted in hyperphagia, obesity and hyperglycaemia. We conclude that CCKVMH neurons are implicated in the regulation of food intake, body weight and glucose homeostasis in the adult brain.

The Neuregulin 1 (NRG1)-ErbB4 signalling pathway is implicated in critical processes for the development and function of neuronal circuits. Post mortem studies have reported that elevated expression of NRG1 type 1 isoform is associated with schizophrenia. Importantly previous behavioural studies in mice that overexpress the NRG1 type 1 isoform (NRG1tg-type-I) have suggested a schizophrenia endophenotype including impairment in the hippocampus-dependent spatial working memory, prepulse inhibition (PPI) of the startle reflex and alterations in the gamma band rhythmogenesis This study aims to reveal the cellular targets of the NRG1-ErbB4 signalling pathway and putative alterations in the function of the hippocampal network in NRG1tg-type-I mice. Immunocytochemical analysis showed that the NRG1 receptor ErbB4 is predominantly localized in interneurons comprising parvalbumin positive (PV) and cholecystokinin (CCK) expressing cells. Comparison of the density of ErbB4-positive cells between the hippocampus of wild type (WT) and NRG1tg-type-I mice suggested that NRG1 over-expression resulted in decreased number of ErbB4 immunopositive hippocampal interneurons. This is consistent with the proposed role of the NRG1-ErbB4 signalling in the migration of GABAergic cells during neurodevelopment and with the NRG1-mediated internalisation of the ErbB4 receptors. CCK- positive cells are a major target of NRG1-ErbB4 signalling, and therefore the NMDA receptor and AMPA receptor components of glutamatergic transmission were analysed in this population of cells by performing whole cell recordings of evoked and miniature excitatory post synaptic currents. Glutamatergic neurotransmission in CCK-positive cells was found to be compromised in the hippocampus of NRG1tg-type-I mice. This change was attributed to hypofunction of NMDA receptors but not AMPA receptors post-synaptically. Next, the inhibitory output of CCK-positive cells to pyramidal cells was examined. Analysis of the optogenetically elicited inhibitory post synaptic currents (IPSCs) did not reveal any changes in the properties of the GABAergic synapse formed by these cells due to NRG1 over-expression Finally, the effects of this NMDA receptor hypofunction in the recurrent inhibition were analysed by performing whole cell recordings during the gamma relevant optogenetic entrainment of the hippocampal network. It was found that the disynaptic inhibition, a key synaptic interaction for the generation of gamma oscillations, depends on the NMDA receptors and was altered in the hippocampus of NRG1tg-type-I mice. Together these data point out a key modulatory role of the NRG1-ErbB4 signalling in the neurodevelopment of cortical microcircuits and a link between ErbB4 and NMDA receptor function with a possible association to schizophrenia pathogenesis.

Transcriptional activation of metallothionein (MT)-encoding genes ( MT ) is regulated during heavy metal induction by short non-identical repeats, termed ‘metal regulatory elements’ ( MRE ), present in multiple imperfect copies in MT promoter regions of eukaryotes. Using mobility shift assays, we have studied the interaction between the human MRE 3 and 4 regions (h MRE 3/4) of the MTIIa promoter and nuclear proteins from uninduced and Cd 2+ -induced HeLa cells, and from Cd 2+ -superinduced H454 cells, a HeLa-derived Cd 2+ -resistant cell isolate which overexpresses h MTIIa after exposure to metal. A specific complex with a similar electrophoretic mobility was formed in all three extracts. Dialysis of the extracts using EDTA inhibited the formation of the complexes, which could be reconstituted only after the addition of Zn 2+ . UV cross-linking analyses of the specific complexes formed by the three nuclear extracts interacting with the h MRE3/4 region revealed that in all of them polypeptides were present having similar electrophoretic mobilities and different molecular masses. Mobility shift assays showed no major differences in the binding of nuclear proteins from induced or uninduced cells. Proposed models of activation of metal-induced MT transcription are discussed.

The expression of metallothionein (MT) and heat shock protein gene families was investigated in normal and in HeLa-derived cadmium-resistant cells, named H454. In the absence of amplification of MT genes H454 cells accumulated elevated concentrations of cadmium ions and synthesized higher levels of MT proteins than unselected HeLa cells. Northern blot analyses revealed higher levels of MT mRNAs in the resistant cells than in wild-type cells after Cd2+and Zn2+exposure. Evaluation of the cytotoxic potential of the different metals confirmed the high resistance to cadmium of the H454 cells. Two proteins of the heat shock family, hsp70 and GRP78, were synthesized in Cd2+-exposed H454 cells at levels comparable to the ones present in Cd2+-treated normal cells. Northern blot analyses of the mRNA levels corresponding to these proteins revealed elevated expression of both hsp70 and GRP78 mRNAs in H454 cells upon exposure to cadmium ions and no response to zinc induction. These data suggest the existence in the H454 cells of a cadmium-specific pathway of regulation of MT and heat shock genes.