Mature Hippocampal Neurons Research Articles

Downregulation of the Polycomb-Associated Methyltransferase Ezh2 during Maturation of Hippocampal Neurons Is Mediated by MicroRNAs Let-7 and miR-124.

Ezh2 is a catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates epigenetic gene silencing through depositing the mark histone H3 lysine 27 trimethylation (H3K27me3) at target genomic sequences. Previous studies have demonstrated that Enhancer of Zeste Homolog 2 (Ezh2) was differentially expressed during maturation of hippocampal neurons; in immature neurons, Ezh2 was abundantly expressed, whereas in mature neurons the expression Ezh2 was significantly reduced. Here, we report that Ezh2 is downregulated by microRNAs (miRs) that are expressed during the hippocampal maturation process. We show that, in mature hippocampal neurons, lethal-7 (let-7) and microRNA-124 (miR-124) are robustly expressed and can target cognate motifs at the 3′-UTR of the Ezh2 gene sequence to downregulate Ezh2 expression. Together, these data demonstrate that the PRC2 repressive activity during hippocampal maturation is controlled through a post-transcriptional mechanism that mediates Ezh2 downregulation in mature neurons.

The soluble neurexin-1\u03b2 ectodomain causes calcium influx and augments dendritic outgrowth and synaptic transmission

Classically, neurexins are thought to mediate synaptic connections through trans interactions with a number of different postsynaptic partners. Neurexins are cleaved by metalloproteases in an activity-dependent manner, releasing the soluble extracellular domain. Here, we report that in both immature (before synaptogenesis) and mature (after synaptogenesis) hippocampal neurons, the soluble neurexin-1β ectodomain triggers acute Ca2+-influx at the dendritic/postsynaptic side. In both cases, neuroligin-1 expression was required. In immature neurons, calcium influx required N-type calcium channels and stimulated dendritic outgrowth and neuronal survival. In mature glutamatergic neurons the neurexin-1β ectodomain stimulated calcium influx through NMDA-receptors, which increased presynaptic release probability. In contrast, prolonged exposure to the ectodomain led to inhibition of synaptic transmission. This secondary inhibition was activity- and neuroligin-1 dependent and caused by a reduction in the readily-releasable pool of vesicles. A synthetic peptide modeled after the neurexin-1β:neuroligin-1 interaction site reproduced the cellular effects of the neurexin-1β ectodomain. Collectively, our findings demonstrate that the soluble neurexin ectodomain stimulates growth of neurons and exerts acute and chronic effects on trans-synaptic signaling involved in setting synaptic strength.

Distribution of Acid Sensing Ion Channels in Axonal Growth Cones and Presynaptic Membrane of Cultured Hippocampal Neurons.

Although acid-sensing ion channels (ASICs) are widely expressed in the central nervous system, their distribution and roles in axonal growth cones remain unclear. In this study, we examined ASIC localization and function in the axonal growth cones of cultured immature hippocampal neurons. Our immunocytochemical data showed that native and overexpressed ASIC1a and ASIC2a are both localized in growth cones of cultured young hippocampal neurons. Calcium imaging and electrophysiological assay results were utilized to validate their function. The calcium imaging test results indicated that the ASICs (primarily ASIC1a) present in growth cones mediate calcium influx despite the addition of voltage-gated Ca2+ channels antagonists and the depletion of intracellular calcium stores. The electrophysiological tests results suggested that a rapid decrease in extracellular pH at the growth cones of voltage-clamped neurons elicits inward currents that were blocked by bath application of the ASIC antagonist amiloride, showing that the ASICs expressed at growth cones are functional. The subsequent immuno-colocalization test results demonstrated that ASIC1a and ASIC2a are both colocalized with Neurofilament-H and Bassoon in mature hippocampal neurons. This finding demonstrated that after reaching maturity, ASIC1a and ASIC2a are both distributed in axons and the presynaptic membrane. Our data reveal the distribution of functional ASICs in growth cones of immature hippocampal neurons and the presence of ASICs in the axons and presynaptic membrane of mature hippocampal neurons, indicating a possible role for ASICs in axonal guidance, synapse formation and neurotransmitter release.

Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner.

The brain-derived neurotrophic factor (BDNF) plays crucial roles in both the developing and mature brain. Moreover, alterations in BDNF levels are correlated with the cognitive impairment observed in several neurological diseases. Among the different therapeutic strategies developed to improve endogenous BDNF levels is the administration of the BDNF-inducing drug Fingolimod, an agonist of the sphingosine-1-phosphate receptor. Fingolimod treatment was shown to rescue diverse symptoms associated with several neurological conditions (i.e., Alzheimer disease, Rett syndrome). However, the cellular mechanisms through which Fingolimod mediates its BDNF-dependent therapeutic effects remain unclear. We show that Fingolimod regulates the dendritic architecture, dendritic spine density and morphology of healthy mature primary hippocampal neurons. Moreover, the application of Fingolimod upregulates the expression of activity-related proteins c-Fos and pERK1/2 in these cells. Importantly, we show that BDNF release is required for these actions of Fingolimod. As alterations in neuronal structure underlie cognitive impairment, we tested whether Fingolimod application might prevent the abnormalities in neuronal structure typical of two neurodevelopmental disorders, namely Rett syndrome and Cdk5 deficiency disorder. We found a significant rescue in the neurite architecture of developing cortical neurons from Mecp2 and Cdkl5 mutant mice. Our study provides insights into understanding the BDNF-dependent therapeutic actions of Fingolimod.

Microtubule‐associated protein 2 mediates induction of long‐term potentiation in hippocampal neurons

Microtubule-associated protein (MAP) 2 has been perceived as a static cytoskeletal protein enriched in neuronal dendritic shafts. Emerging evidence indicates dynamic functions for various MAPs in activity-dependent synaptic plasticity. However, it is unclear how MAP2 is associated with synaptic plasticity mechanisms. Here, we demonstrate that specific silencing of high-molecular-weight MAP2 in vivo abolished induction of long-term potentiation (LTP) in the Schaffer collateral pathway of CA1 pyramidal neurons and in vitro blocked LTP-induced surface delivery of AMPA receptors and spine enlargement. In mature hippocampal neurons, we observed rapid translocation of a subpopulation of MAP2, present in dendritic shafts, to spines following LTP stimulation. Time-lapse confocal imaging showed that spine translocation of MAP2 was coupled with LTP-induced spine enlargement. Consistently, immunogold electron microscopy revealed that LTP stimulation of the Schaffer collateral pathway promoted MAP2 labeling in spine heads of CA1 neurons. This translocation depended on NMDA receptor activation and Ras-MAPK signaling. Furthermore, LTP stimulation led to an increase in surface-expressed AMPA receptors specifically in the neurons with MAP2 spine translocation. Altogether, this study indicates a novel role for MAP2 in LTP mechanisms and suggests that MAP2 participates in activity-dependent synaptic plasticity in mature hippocampal networks.

Mitochondrial-associated impairments of temozolomide on neural stem/progenitor cells and hippocampal neurons

Primary brain tumor patients often experience neurological, cognitive, and depressive symptoms that profoundly affect quality of life. The DNA alkylating agent, temozolomide (TMZ), along with radiation therapy forms the standard of care for glioblastoma (GBM) – the most common and aggressive of all brain cancers. Numerous studies have reported that TMZ disrupts hippocampal neurogenesis and causes spatial learning deficits in rodents; however, the effect of TMZ on mature hippocampal neurons has not been addressed. In this study, we examined the mitochondrial-mediated mechanisms involving TMZ-induced neural damage in primary rat neural stem/progenitor cells (NSC) and hippocampal neurons. TMZ inhibited mtDNA replication and transcription of mitochondrial genes (ND1 and Cyt b) in NSC by 24 h, whereas the effect of TMZ on neuronal mtDNA transcription was less pronounced. Transmission electron microscopy imaging revealed mitochondrial degradation in TMZ-treated NSC. Acute TMZ exposure (4 h) caused a rapid reduction in dendritic branching and loss of postsynaptic density-95 (PSD95) puncta on dendrites. Longer TMZ exposure impaired mitochondrial respiratory activity, increased oxidative stress, and induced apoptosis in hippocampal neurons. The presented findings suggest that NSC may be more vulnerable to TMZ than hippocampal neurons upon acute exposure; however long-term TMZ exposure results in neuronal mitochondrial respiratory dysfunction and dendritic damage, which may be associated with delayed cognitive impairments.

Neuronal O-GlcNAcylation Improves Cognitive Function in the Aged Mouse Brain.

SUMMARYMounting evidence in animal models indicates potential for rejuvenation of cellular and cognitive functions in the aging brain. However, the ability to utilize this potential is predicated on identifying molecular targets that reverse the effects of aging in vulnerable regions of the brain, such as the hippocampus. The dynamic post-translational modification O-linked N-Acetylglucosamine (O-GlcNAc) has emerged as an attractive target for regulating aging-specific synaptic alterations as well as neurodegeneration. While speculation exists about the role of O-GlcNAc in neurodegenerative conditions, such as Alzheimer’s disease, its role in physiological brain aging remains largely unexplored. Here, we report that countering age-related decreased O-GlcNAc transferase (OGT) expression and O-GlcNAcylation ameliorates cognitive impairments in aged mice. Mimicking an aged condition in young adults by abrogating OGT, using a temporally controlled neuron-specific conditional knockout mouse model, recapitulated cellular and cognitive features of brain aging. Conversely, overexpressing OGT in mature hippocampal neurons using a viral-mediated approach enhanced associative fear memory in young adult mice. Excitingly, in aged mice overexpressing neuronal OGT in the aged hippocampus rescued in part age-related impairments in spatial learning and memory as well as associative fear memory. Our data identify O-GlcNAcylaton as a key molecular mediator promoting cognitive rejuvenation.

The Applications of Lattice Light-sheet Microscopy for Functional Volumetric Imaging of Hippocampal Neurons in a Three-Dimensional Culture System.

The characterization of individual cells in three-dimensions (3D) with very high spatiotemporal resolution is crucial for the development of organs-on-chips, in which 3D cell cultures are integrated with microfluidic systems. In this study, we report the applications of lattice light-sheet microscopy (LLSM) for monitoring neuronal activity in three-dimensional cell culture. We first established a 3D environment for culturing primary hippocampal neurons by applying a scaffold-based 3D tissue engineering technique. Fully differentiated and mature hippocampal neurons were observed in our system. With LLSM, we were able to monitor the behavior of individual cells in a 3D cell culture, which was very difficult under a conventional microscope due to strong light scattering from thick samples. We demonstrated that our system could study the membrane voltage and intracellular calcium dynamics at subcellular resolution in 3D under both chemical and electrical stimulation. From the volumetric images, it was found that the voltage indicators mainly resided in the cytosol instead of the membrane, which cannot be distinguished using conventional microscopy. Neuronal volumetric images were sheet scanned along the axial direction and recorded at a laser exposure of 6 ms, which covered an area up to 4800 μm2, with an image pixel size of 0.102 μm. When we analyzed the time-lapse volumetric images, we could quantify the voltage responses in different neurites in 3D extensions.

Mechanisms underpinning AMP-activated protein kinase-related effects on behavior and hippocampal neurogenesis in an animal model of depression

Adenosine monophosphate-activated protein kinase (AMPK) is critical for whole-body energy metabolism regulation. Recent studies have suggested that physical exercise ameliorates depressive-like behaviors via AMPK activation; however, the underlying mechanism is unclear. Here, we examined the effects and underlying mechanisms of AMPK activation on depressive-like behavior in olfactory bulbectomized (OBX) mice. We treated OBX mice with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleotide (AICAR) on the 7th or 14th day after bilateral bulbectomy and evaluated depressive-like behavior using the tail-suspension test (TST) and forced swimming test (FST) on the 21st day. The expression of phosphorylated AMPK, protein kinase C ζ (PKCζ), nuclear factor-kappa B (NF-κB), brain-derived neurotrophic factor (BDNF), and cAMP response element-binding protein (CREB) in the hippocampus was assessed by western blotting. Hippocampal neurogenesis and localization of AMPK and phosphorylated NF-κB were examined by immunohistochemistry. Chronic AICAR treatment suppressed the prolonged immobility of OBX mice in the TST and FST, and increased the levels of phosphorylated AMPK, PKCζ, NF-κB, CREB, and BDNF. Hippocampal neurogenesis in OBX mice was promoted by chronic AICAR treatment. Co-administration of AICAR with the PKCζ inhibitor or the neurotrophic tyrosine kinase receptor type 2 (TrkB) antagonist, ANA-12, inhibited these effects. Phosphorylated AMPK was detected in mature and immature hippocampal neurons and microglia, while phosphorylated NF-κB was detected only in neurons in AICAR-treated OBX mice. These data indicate that AMPK activation produces anti-depressant effects, which are mediated by elevated hippocampal neurogenesis potentially via PKCζ/NF-κB/BDNF/TrkB/CREB signaling in neurons.

Midazolam prevents sevoflurane-induced death in hippocampal neurons

Sevoflurane is a widely used anesthetics in surgery and considered as a safe reagent for clinical use. However, recent studies demonstrated that sevoflurane has a neurotoxic effect in central nervous system. Thus, finding ways to alleviate the side effect of sevoflurane is of importance. In this study, we identified the neuroprotective role of midazolam in hippocampal neurons. Midazolam treatment could alleviate the neuronal death and promote the neuronal maturation in hippocampal neurons in vitro. In vivo studies demonstrated that midazolam injection could improve behavioral deficit in sevoflurane-exposed animals. The anti-apoptotic function of midazolam in sevoflurane-exposed neurons was mediated by ERK signaling. Collectively, we elucidated a new role of midazolam in preventing hippocampal neuronal death from sevoflurane exposure, potentially providing a new strategy to resist the neurotoxicity in the clinical application of sevoflurane.

Kalirin-7 prevents dendritic spine dysgenesis induced by amyloid beta-derived oligomers.

Synapse degeneration and dendritic spine dysgenesis are believed to be crucial early steps in Alzheimer's disease (AD), and correlate with cognitive deficits in AD patients. Soluble amyloid beta (Aβ)-derived oligomers, also termed Aβ-derived diffusible ligands (ADDLs), accumulate in the brain of AD patients and play a crucial role in AD pathogenesis. ADDLs bind to mature hippocampal neurons, induce structural changes in dendritic spines and contribute to neuronal death. However, mechanisms underlying structural and toxic effects are not fully understood. Here, we report that ADDLs bind to cultured mature cortical pyramidal neurons and induce spine dysgenesis. ADDL treatment induced the rapid depletion of kalirin-7, a brain-specific guanine-nucleotide exchange factor for the small GTPase Rac1, from spines. Kalirin-7 is a key regulator of dendritic spine morphogenesis and maintenance in forebrain pyramidal neurons and here we show that overexpression of kalirin-7 prevents ADDL-induced spine degeneration.Taken together, our results suggest that kalirin-7 may play a role in the early events leading to synapse degeneration, and its pharmacological activation may prevent or delay synapse pathology in AD.

EphA4 Regulates Hippocampal Neural Precursor Proliferation in the Adult Mouse Brain by d-Serine Modulation of N-Methyl-d-Aspartate Receptor Signaling.

The hippocampal dentate gyrus (DG) is a major region of the adult rodent brain in which neurogenesis occurs throughout life. The EphA4 receptor, which regulates neurogenesis and boundary formation in the developing brain, is also expressed in the adult DG, but whether it regulates adult hippocampal neurogenesis is not known. Here, we show that, in the adult mouse brain, EphA4 inhibits hippocampal precursor cell proliferation but does not affect precursor differentiation or survival. Genetic deletion or pharmacological inhibition of EphA4 significantly increased hippocampal precursor proliferation in vivo and in vitro, by blocking EphA4 forward signaling. EphA4 was expressed by mature hippocampal DG neurons but not neural precursor cells, and an EphA4 antagonist, EphA4-Fc, did not activate clonal cultures of precursors until they were co-cultured with non-precursor cells, indicating an indirect effect of EphA4 on the regulation of precursor activity. Supplementation with d-serine blocked the increased precursor proliferation induced by EphA4 inhibition, whereas blocking the interaction between d-serine and N-methyl-d-aspartate receptors (NMDARs) promoted precursor activity, even at the clonal level. Collectively, these findings demonstrate that EphA4 indirectly regulates adult hippocampal precursor proliferation and thus plays a role in neurogenesis via d-serine-regulated NMDAR signaling.

TAT-Ngn2 Enhances Cognitive Function Recovery and Regulates Caspase-Dependent and Mitochondrial Apoptotic Pathways After Experimental Stroke.

Neurogenin-2 (Ngn2) is a basic helix-loop-helix (bHLH) transcription factor that contributes to the identification and specification of neuronal fate during neurogenesis. In our previous study, we found that Ngn2 plays an important role in alleviating neuronal apoptosis, which may be viewed as an attractive candidate target for the treatment of cerebral ischemia. However, novel strategies require an understanding of the function and mechanism of Ngn2 in mature hippocampal neurons after global cerebral ischemic injury. Here, we found that the expression of Ngn2 decreased in the hippocampus after global cerebral ischemic injury in mice and in primary hippocampal neurons after oxygen glucose deprivation (OGD) injury. Then, transactivator of transcription (TAT)-Ngn2, which was constructed by fusing a TAT domain to Ngn2, was effectively transported and incorporated into hippocampal neurons after intraperitoneal (i.p.) injection and enhanced cognitive functional recovery in the acute stage after reperfusion. Furthermore, TAT-Ngn2 alleviated hippocampal neuronal damage and apoptosis, and inhibited the cytochrome C (CytC) leak from the mitochondria to the cytoplasm through regulating the expression levels of brain-derived neurotrophic factor (BDNF), phosphorylation tropomyosin-related kinase B (pTrkB), Bcl-2, Bax and cleaved caspase-3 after reperfusion injury in vivo and in vitro. These findings suggest that the downregulation of Ngn2 expression may have an important role in triggering brain injury after ischemic stroke and that the neuroprotection of TAT-Ngn2 against stroke might involve the modulation of BDNF-TrkB signaling that regulates caspase-dependent and mitochondrial apoptotic pathways, which may be an attractive therapeutic strategy for cerebral ischemic injury.

Super-resolving botulinum neurotoxin type A molecules: from surface landing to internalization in synaptic vesicles

Botulinum neurotoxin type-A (BoNT/A) is internalized into motor nerve terminals as part of its intoxication strategy to incapacitate nerve-muscle communication. The “dual receptor” model explained how BoNT/A initially interacts with GT1b gangliosides, thereby concentrating the toxin on the presynaptic membrane to foster the subsequent interaction with a proteinaceous co-receptor SV2 which triggers receptor-mediated endocytosis. I will revisit the “dual receptor” concept using two single-molecule imaging strategies,1-3 allowing tracking of single Atto647N-labeled BoNT/A molecules upon (i) landing on the plasma membrane (by uPAINT) and (ii) internalization in synaptic vesicles (by sdTIM) of living mature hippocampal neurons. With 30 to 40 nm localization precision, we revealed that once internalized in synaptic vesicles, Atto647N-tagged BoNT/A exhibits a markedly lower mobility than on the plasma membrane. I will discuss how individual genetic inactivation of the GT1b and SV2 binding sites of the neurotoxin affects the diffusion states of BoNT/A mutants on the plasma membrane and axonal trafficking. Single neurotoxin super-resolution imaging uncovers an updated dual receptor model taking into consideration the diffusive patterns generated by each of the co-receptors and leading to defined nanoscale dynamic organizations at key steps of the intoxication journey.

Chronic Treatment with Combined Chemotherapeutic Agents Affects Hippocampal Micromorphometry and Function in Mice, Independently of Neuroinflammation.

Chemotherapeutic agents induce long-term side effects, including cognitive impairment and mood disorders, particularly in breast cancer survivors who have undergone chemotherapy. However, the precise mechanisms underpinning chemotherapy-induced hippocampal dysfunction remain unknown. In this study, we investigated the detrimental effects of chronic treatment with a combination of adriamycin and cyclophosphamide (AC) on the neuronal architecture and functions of the hippocampi of female C57BL/6 mice. After chronic AC administration, mice showed memory impairment (measured using a novel object recognition memory task) and depression-like behavior (measured using the tail suspension test and forced swim test). According to Golgi staining, chronic AC treatment significantly reduced the total dendritic length, ramification, and complexity as well as spine density and maturation in hippocampal neurons in a sub-region-specific manner. Additionally, the AC combination significantly reduced adult neurogenesis, the extent of the vascular network, and the levels of hippocampal angiogenesis-related factors. However, chronic AC treatment did not increase the levels of inflammation-related signals (microglial or astrocytic distribution, or the levels of pro-inflammatory cytokines or M1/M2 macrophage markers). Thus, chronic AC treatment changed the neuronal architecture of the adult hippocampus, possibly by reducing neurogenesis and the extent of the vasculature, independently of neuroinflammation. Such detrimental changes in micromorphometric parameters may explain the hippocampal dysfunction observed after cancer chemotherapy.

Assessment of systemic administration of PEGylated IGF-1 in a mouse model of traumatic brain injury.

Traumatic brain injury can result in lasting cognitive dysfunction due to degeneration of mature hippocampal neurons as well as the loss of immature neurons within the dentate gyrus. While endogenous neurogenesis affords a partial recovery of the immature neuron population, hippocampal neurogenesis may be enhanced through therapeutic intervention. Insulin-like growth factor-1 (IGF-1) has the potential to improve cognitive function and promote neurogenesis after TBI, but its short half-life in the systemic circulation makes it difficult to maintain a therapeutic concentration. IGF-1 modified with a polyethylene glycol moiety (PEG-IGF-1) exhibits improved stability and half-life while retaining its ability to enter the brain from the periphery, increasing its viability as a translational approach. The goal of this study was to evaluate the ability of systemic PEG-IGF-1 administration to attenuate acute neuronal loss and stimulate the recovery of hippocampal immature neurons in brain-injured mice. In a series of studies utilizing a well-established contusion brain injury model, PEG-IGF-1 was administered subcutaneously after injury. Serum levels of PEG were verified using ELISA and histological staining was used to investigate numbers of degenerating neurons and cortical contusion size at 24 h after injury. Immunofluorescent staining was used to evaluate numbers of immature neurons at 10 d after injury. Although subcutaneous injections of PEG-IGF-1 increased serum IGF-1 levels in a dose-dependent manner, no effects were observed on cortical contusion size, neurodegeneration within the dentate gyrus, or recovery of hippocampal immature neuron numbers. In contrast to its efficacy in rodent models of neurodegenerative diseases, PEG- IGF-1 was not effective in ameliorating early neuronal loss after contusion brain trauma.

Histone deacetylase 4 shapes neuronal morphology via a mechanism involving regulation of expression of vascular endothelial growth factor D

Nucleo-cytoplasmic shuttling of class IIa histone deacetylases (i.e HDAC4, -5, -7, and -9) is a synaptic activity- and nuclear calcium-dependent mechanism important for epigenetic regulation of signal-regulated gene expression in hippocampal neurons. HDAC4 in particular has been linked to the regulation of genes important for both synaptic structure and plasticity. Here, using a constitutively nuclear-localized, dominant-active variant of HDAC4 (HDAC4 3SA), we demonstrate that HDAC4 accumulation in the nucleus severely reduces both the length and complexity of dendrites of cultured mature hippocampal neurons, but does not affect the number of dendritic spines. This phenomenon appeared to be specific to HDAC4, as increasing the expression of HDAC3 or HDAC11, belonging to class I and class IV HDACs, respectively, did not alter dendritic architecture. We also show that HDAC4 3SA decreases the expression of vascular endothelial growth factor D (VEGFD), a key protein required for the maintenance of dendritic arbors. The expression of other members of the VEGF family and their receptors was not affected by the nuclear accumulation of HDAC4. VEGFD overexpression or administration of recombinant VEGFD, but not VEGFC, the closest VEGFD homologue, rescued the impaired dendritic architecture caused by the nuclear-localized HDAC4 variant. These results identify HDAC4 as an epigenetic regulator of neuronal morphology that controls dendritic arborization via the expression of VEGFD.

P.1.010 - Brain-derived neurotrophic factor enhances glutamic acid decarboxylase and perineuronal net maturation in mouse primary cortical cultures

Schizophrenia is a debilitating mental illness which affects approximately 1% of the global population, and is characterised by positive (e.g. hallucinations) and negative (e.g. anhedonia) symptoms, combined with cognitive deficits. Evidence suggests diminished cortical inhibitory drive as a major contributing factor in schizophrenia pathophysiology. Reductions in glutamic acid decarboxylase (GAD65/67) and deficits in the extracellular matrix structures, perineuronal nets (PNNs), which enwrap parvalbumin-containing interneurons have been observed in patients [1,2]. Members of the c-Jun N-Terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) and their upstream kinase MKK7 have been implicated in neocortical development. Interestingly, MAP2K7 (encoding MKK7) has been implicated as a candidate risk gene for schizophrenia [3]. However, the impact of disrupted MAP2K7-JNK signalling on GABAergic interneuron development remains elusive. Reduced levels of brain-derived neurotrophic factor (BDNF) have also been found in patients [4]. Previous research highlights BDNF as a promoter of GABAergic maturation in hippocampal neurons [5]. Consequently, altered BDNF activity could result in abnormal cortical circuitry development and contribute to the cortical neuronal dysfunction underlying schizophrenia. Here we study the effects of BDNF on the development of primary cortical GABAergic interneurons and surrounding PNNs along with the possible involvement of JNK signalling. Mouse primary cortical neuronal cultures were treated at 7 days in vitro with either vehicle, BDNF (50 ng/ml), JNK inhibitor (SP600125, 5 μM) or BDNF+JNK inhibitor. Subsequently, immunofluorescent-cytochemistry (ICC) was performed. PNN development was assessed using Wisteria floribunda agglutinin (WFA) lectin which labels interneuron PNNs. Cultures were double-labelled with GAD65/67. Normalised intensity data (in relative optical density) were expressed as a percentage of vehicle and analysed via three-way ANOVA with Tukey post-hoc multiple comparisons with results considered significant if p Analysis of WFA staining intensity revealed significant main effects of BDNF (F1, 982 = 45.46, p Our data demonstrate that BDNF increased GAD65/67 levels and PNN density in developing cortical interneurons. Our data suggest this enhancing effect of BDNF on GAD65/67 and PNN maturation is dependent on JNK signalling. Given evidence of reduced BDNF levels in schizophrenia patients, we speculate that impairments in MAP2K7-JNK signalling may be linked with functional BDNF deficits in schizophrenia and that BDNF disruption could be involved in disorder aetiology and pathophysiology.

The female epilepsy protein PCDH19 is a new GABAAR-binding partner that regulates GABAergic transmission as well as migration and morphological maturation of hippocampal neurons.

The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABAA receptor (GABAAR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABAAR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.

MicroRNA-16 targets mRNA involved in neurite extension and branching in hippocampal neurons during presymptomatic prion disease

The mechanisms that lead to neuronal death in neurodegenerative diseases are poorly understood. Prion diseases, like many more common disorders such as Alzheimer's and Parkinson's diseases, are characterized by the progressive accumulation of misfolded disease-specific proteins. The earliest changes observed in brain tissue include a reduction in synaptic number and retraction of dendritic spines, followed by reduced length and branching of neurites. These pathologies are observable during presymptomatic stages of disease and are accompanied by altered expression of transcripts that include miRNAs. Here we report that miR-16 localized within hippocampal CA1 neurons is increased during early prion disease. Modulating miR-16 expression in mature murine hippocampal neurons by expression from a lentivirus, thus mimicking the modest increase seen in vivo, was found to induce neurodegeneration. This was characterized by retraction of neurites and reduced branching. We performed immunoprecipitation of the miR-16 enriched RISC complex, and identified associated transcripts from the co-immunoprecipitated RNA (Ago2 RIP-Chip). These transcripts were enriched with predicted binding sites for miR-16, including the validated miR-16 targets APP and BCL2, as well as numerous novel targets. In particular, genes within the neurotrophin receptor mediated MAPK/ERK pathway were potentially regulated by miR-16; including TrkB (NTRK2), MEK1 (MAP2K1) and c-Raf (RAF). Increased miR-16 expression in neurons during presymptomatic prion disease and reduction in proteins involved in MAPK/ERK signaling represents a possible mechanism by which neurite length and branching are decreased during early stages of disease.