Hydroxychloroquine Mitigates the Production of 8-Isoprostane and Improves Vascular Dysfunction: Implications for Treating Preeclampsia.

In preeclampsia, widespread maternal endothelial dysfunction is often secondary to excessive generation of placental-derived anti-angiogenic factors, including soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), along with proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and activin A, understanding of which offers potential opportunities for the development of novel therapies. The antimalarial hydroxychloroquine is an anti-inflammatory drug improving endothelial homeostasis in lupus. It has not been explored as to whether it can improve placental and endothelial function in preeclampsia. In this in vitro study, term placental explants were used to assess the effects of hydroxychloroquine on placental production of sFlt-1, sEng, TNF-α, activin A, and 8-isoprostane after exposure to hypoxic injury or oxidative stress. Similarly, human umbilical vein endothelial cells (HUVECs) were used to assess the effects of hydroxychloroquine on in vitro markers of endothelial dysfunction. Hydroxychloroquine had no effect on the release of sFlt-1, sEng, TNF-α, activin A, or 8-isoprostane from placental explants exposed to hypoxic injury or oxidative stress. However, hydroxychloroquine mitigated TNF-α-induced HUVEC production of 8-isoprostane and Nicotinanamide adenine dinucleotide phosphate (NADPH) oxidase expression. Hydroxychloroquine also mitigated TNF-α and preeclamptic serum-induced HUVEC monolayer permeability and rescued the loss of zona occludens protein zona occludens 1 (ZO-1). Although hydroxychloroquine had no apparent effects on trophoblast function, it may be a useful endothelial protectant in women presenting with preeclampsia.

Sulforaphane improves endothelial function and reduces placental oxidative stress in vitro.

INTRODUCTION: The maternal endothelial dysfunction characteristic of preeclampsia arises, in part, from excessive placental production of anti-angiogenic factors, including soluble Flt-1, soluble endoglin and activin A, inducing oxidative stress. We assessed whether the antioxidant and NRF2-activator sulforaphane could mitigate endothelial and trophoblast dysfunction in vitro. METHODS: We induced dysfunction in human umbilical vein endothelial cells (HUVECs) with TNF-α, assessing endothelial activation and dysfunction (endothelin-1, vascular cell adhesion molecule; VCAM1, intracellular adhesion molecule; ICAM1, e-selectin and endothelial permeability) in the presence or absence of sulforaphane. We also assessed the effects of sulforaphane in mitigating hypoxic and hyperoxic injury in term placental explants by measuring secretion of anti-angiogenic factors. To assess the role of NRF2 we silenced NRF2 in HUVECs and primary trophoblast cells. RESULTS: Sulforaphane reduced TNF-α mediated HUVEC secretion of endothelin-1, VCAM1, ICAM1 and E-selectin, and prevented increased endothelial permeability. In placental explants, sulforaphane reduced the secretion of soluble Flt-1, soluble endoglin and activin A. Sulforaphane induced activation and nuclear translocation of NRF2 in HUVECs, inducing heme oxygenase 1. NRF2 silencing blocked some but not all of sulforaphane's effects in HUVECs. NRF2 silencing did not prevent sulforaphane's inhibition of trophobast secretion of soluble Flt-1 or activin A. CONCLUSION: In reducing placental and endothelial oxidative stress, sulforaphane may offer a new adjuvant therapeutic approach for the treatment of preeclampsia.

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation.

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair.

Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell-based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell-derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell-derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibrosis. Exosomes were isolated and evaluated for their protein and miRNA cargo. Direct effects of hAEC Exo on immune cell function, including macrophage polarization, phagocytosis, neutrophil myeloperoxidase activity and T cell proliferation and uptake, were measured. Their impact on immune response, histological outcomes, and bronchioalveolar stem cell (BASC) response was assessed in vivo following bleomycin challenge in young and aged mice. hAEC Exo carry protein cargo enriched for MAPK signaling pathways, apoptotic and developmental biology pathways and miRNA enriched for PI3K-Akt, Ras, Hippo, TGFß, and focal adhesion pathways. hAEC Exo polarized and increased macrophage phagocytosis, reduced neutrophil myeloperoxidases, and suppressed T cell proliferation directly. Intranasal instillation of 10 µg hAEC Exo 1 day following bleomycin challenge reduced lung inflammation, while treatment at day 7 improved tissue-to-airspace ratio and reduced fibrosis. Administration of hAEC Exo coincided with the proliferation of BASC. These effects were reproducible in bleomycin-challenged aged mice. The paracrine effects of hAECs can be largely attributed to their exosomes and exploitation of hAEC Exo as a therapy for IPF should be explored further. Stem Cells Translational Medicine 2018;7:180-196.

Real-Time Blood Pressure Recording Using Radiotelemetry in a Rat Model of Preeclampsia.

Radiotelemetry is increasingly being recognized not just as the gold standard but a necessity for validation of gestational hypertension seen in preeclampsia. Here we describe radiotelemetry probe implantation into the descending aorta of Sprague-Dawley rats to allow real-time blood pressure recording over the entire gestational period. This is a valuable tool to be able to track changes in maternal blood pressure throughout gestation and the efficacy of novel therapeutic agents in controlling hypertension.

Formyl peptide receptor-2 is decreased in foetal growth restriction and contributes to placental dysfunction.

STUDY QUESTION: What is the association between placental formyl peptide receptor 2 (FPR2) and trophoblast and endothelial functions in pregnancies affected by foetal growth restriction (FGR)? SUMMARY ANSWER: Reduced FPR2 placental expression in idiopathic FGR results in significantly altered trophoblast differentiation and endothelial function in vitro. WHAT IS KNOWN ALREADY: FGR is associated with placental insufficiency, where defective trophoblast and endothelial functions contribute to reduced feto-placental growth. STUDY DESIGN, SIZE, DURATION: The expression of FPR2 in placental tissues from human pregnancies complicated with FGR was compared to that in gestation-matched uncomplicated control pregnancies (n = 25 from each group). Fpr2 expression was also determined in placental tissues obtained from a murine model of FGR (n = 4). The functional role of FPR2 in primary trophoblasts and endothelial cells in vitro was assessed in diverse assays in a time-dependent manner. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placentae from third-trimester pregnancies complicated by idiopathic FGR (n = 25) and those from gestation-matched pregnancies with appropriately grown infants as controls (n = 25) were collected at gestation 27-40 weeks. Placental tissues were also collected from a spontaneous CBA/CaH × DBA/2 J murine model of FGR. Placental FPR2/Fpr2 mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence FPR2 expression in primary trophoblasts and in human umbilical vein endothelial cells (HUVEC), and the quantitation of cytokines, chemokines and apoptosis was performed following a cDNA array analyses. Functional effects of trophoblast differentiation were measured using HCGB/ß-hCG and syncytin-2 expression as well as markers of apoptosis, tumour protein 53 (TP53), caspase 8, B cell lymphoma 2 (BCL2) and BCL associated X (BAX). Endothelial function was assessed by proliferation, network formation and permeability assays. MAIN RESULTS AND THE ROLE OF CHANCE: Placental FPR2/Fpr2 expression was significantly decreased in FGR placentae (n = 25, P < 0.05) as well as in murine FGR placentae compared to controls (n = 4, P < 0.05). FPR2 siRNA (siFPR2) in term trophoblasts significantly increased differentiation markers, HCGB and syncytin-2; cytokines, interleukin (IL)-6, CXCL8; and apoptotic markers, TP53, caspase 8 and BAX, but significantly reduced the expression of the chemokines CXCL12 and its receptors CXCR4 and CXCR7; CXCL16 and its receptor, CXCR6; and cytokine, IL-10, compared with control siRNA (siCONT). Treatment of HUVECs with siFPR2 significantly reduced proliferation and endothelial tube formation, but significantly increased permeability of HUVECs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Reduced expression of placental FPR2/Fpr2 was observed in the third trimester at delivery after development of FGR, suggesting that FPR2 is associated with FGR pregnancies. However, there is a possibility that the decreased placental FPR2 observed in FGR may be a consequence rather than a cause of FGR, although our in vitro functional analyses using primary trophoblasts and endothelial cells suggest that FPR2 may have a direct or indirect regulatory role on trophoblast differentiation and endothelial function in FGR. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study linking placental FPR2 expression with changes in the trophoblast and endothelial functions that may explain the placental insufficiency observed in FGR. STUDY FUNDING/COMPETING INTERESTS: P.M. and P.R.E. received funding from the Australian Institute of Musculoskeletal Science, Western Health, St. Albans, Victoria 3021, Australia. M.L. is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; Grant no. 1047025). Monash Health is supported by the Victorian Government's Operational Infrastructure Support Programme. The authors declare that there is no conflict of interest in publishing this work.

Resveratrol mitigates trophoblast and endothelial dysfunction partly via activation of nuclear factor erythroid 2-related factor-2.

INTRODUCTION: Maternal endothelial dysfunction underlying preeclampsia arises from excessive placental release of anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt1), soluble endoglin (sEng) and activin A. Resveratrol, an activator of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, mediates the gene expression of antioxidant and vasoprotective factors that may counter the endothelial damage imposed by these anti-angiogenic factors. The objective of this study was to assess whether resveratrol could reduce placental oxidative stress and production of anti-angiogenic factors in vitro and/or improve in vitro markers of endothelial dysfunction via Nrf2 activation. METHOD: We used in vitro term placental explants to assess the effects of resveratrol on placental oxidative stress and production of sFlt1, sEng and activin A. Using human umbilical vein endothelial cells we investigated the effects of resveratrol on markers of in vitro endothelial dysfunction, including the expression of intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), E-selectin and endothelin-1, and endothelial permeability. To confirm that resveratrol mediated its effects via Nrf2, we examined the impact of resveratrol on the same in vitro markers of endothelial and placental dysfunction following Nrf2 knockdown. RESULTS: Resveratrol significantly decreased placental oxidative stress and the production of sFlt1 and activin A. Resveratrol significantly mitigated tumor necrosis factor-α stimulated endothelial expression of ICAM1, VCAM1, E-selectin and endothelin-1 and prevented an increase in endothelial monolayer permeability. Nrf2 knockdown abolished some of the protective effects of resveratrol on endothelial cells, but not in primary trophoblast cells. CONCLUSION: Features of placental and endothelial dysfunction characteristic of preeclampsia are improved by resveratrol in vitro, partially via the modulation of Nrf2.

The Human Amnion Epithelial Cell Secretome Decreases Hepatic Fibrosis in Mice with Chronic Liver Fibrosis.

Background: Hepatic stellate cells (HSCs) are the primary collagen-secreting cells in the liver. While HSCs are the major cell type involved in the pathogenesis of liver fibrosis, hepatic macrophages also play an important role in mediating fibrogenesis and fibrosis resolution. Previously, we observed a reduction in HSC activation, proliferation, and collagen synthesis following exposure to human amnion epithelial cells (hAEC) and hAEC-conditioned media (hAEC-CM). This suggested that specific factors secreted by hAEC might be effective in ameliorating liver fibrosis. hAEC-derived extracellular vesicles (hAEC-EVs), which are nanosized (40-100 nm) membrane bound vesicles, may act as novel cell-cell communicators. Accordingly, we evaluated the efficacy of hAEC-EV in modulating liver fibrosis in a mouse model of chronic liver fibrosis and in human HSC. Methods: The hAEC-EVs were isolated and characterized. C57BL/6 mice with CCl4-induced liver fibrosis were administered hAEC-EV, hAEC-CM, or hAEC-EV depleted medium (hAEC-EVDM). LX2 cells, a human HSC line, and bone marrow-derived mouse macrophages were exposed to hAEC-EV, hAEC-CM, and hAEC-EVDM. Mass spectrometry was used to examine the proteome profile of each preparation. Results: The extent of liver fibrosis and number of activated HSCs were reduced significantly in CCl4-treated mice given hAEC-EVs, hAEC-CM, and hAEC EVDM compared to untreated controls. Hepatic macrophages were significantly decreased in all treatment groups, where a predominant M2 phenotype was observed. Human HSCs cultured with hAEC-EV and hAEC-CM displayed a significant reduction in collagen synthesis and hAEC-EV, hAEC-CM, and hAEC-EVDM altered macrophage polarization in bone marrow-derived mouse macrophages. Proteome analysis showed that 164 proteins were unique to hAEC-EV in comparison to hAEC-CM and hAEC-EVDM, and 51 proteins were co-identified components with the hAEC-EV fraction. Conclusion: This study provides novel data showing that hAEC-derived EVs significantly reduced liver fibrosis and macrophage infiltration to an extent similar to hAEC-EVDM and hAEC-CM. hAEC-EV-based therapy may be a potential therapeutic option for liver fibrosis.

Human amnion cells reverse acute and chronic pulmonary damage in experimental neonatal lung injury.

BACKGROUND: Despite advances in neonatal care, bronchopulmonary dysplasia (BPD) remains a significant contributor to infant mortality and morbidity. While human amnion epithelial cells (hAECs) have shown promise in small and large animal models of BPD, there is scarce information on long-term benefit and clinically relevant questions surrounding administration strategy remain unanswered. In assessing the therapeutic potential of hAECs, we investigated the impact of cell dosage, administration routes and timing of treatment in a pre-clinical model of BPD. METHODS: Lipopolysaccharide was introduced intra-amniotically at day 16 of pregnancy prior to exposure to 65% oxygen (hyperoxia) at birth. hAECs were administered either 12 hours (early) or 4 days (late) after hyperoxia commenced. Collective lung tissues were subjected to histological analysis, multikine ELISA for inflammatory cytokines, FACS for immune cell populations and 3D lung stem cell culture at neonatal stage (postnatal day 7 and 14). Invasive lung function test and echocardiography were applied at 6 and 10 weeks of age. RESULTS: hAECs improved the tissue-to-airspace ratio and septal crest density in a dose-dependent manner, regardless of administration route. Early administration of hAECs, coinciding with the commencement of postnatal hyperoxia, was associated with reduced macrophages, dendritic cells and natural killer cells. This was not the case if hAECs were administered when lung injury was established. Fittingly, early hAEC treatment was more efficacious in reducing interleukin-1ß, tumour necrosis factor alpha and monocyte chemoattractant protein-1 levels. Early hAEC treatment was also associated with reduced airway hyper-responsiveness and normalisation of pressure-volume loops. Pulmonary hypertension and right ventricle hypertrophy were also prevented in the early hAEC treatment group, and this persisted until 10 weeks of age. CONCLUSIONS: Early hAEC treatment appears to be advantageous over late treatment. There was no difference in efficacy between intravenous and intratracheal administration. The benefits of hAEC administration resulted in long-term improvements in cardiorespiratory function.

Cell Death in the Developing Brain after Hypoxia-Ischemia.

Perinatal insults such as hypoxia-ischemia induces secondary brain injury. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. The cell death mechanisms have been shown to be quite different in the developing brain compared to that in the adult. The aim of this review is update on what cell death mechanisms that are operating particularly in the setting of the developing CNS. In response to mild stress stimuli a number of compensatory mechanisms will be activated, most often leading to cell survival. Moderate-to-severe insults trigger regulated cell death. Depending on several factors such as the metabolic situation, cell type, nature of the stress stimulus, and which intracellular organelle(s) are affected, the cell undergoes apoptosis (caspase activation) triggered by BAX dependent mitochondrial permeabilzation, necroptosis (mixed lineage kinase domain-like activation), necrosis (via opening of the mitochondrial permeability transition pore), autophagic cell death (autophagy/Na+, K+-ATPase), or parthanatos (poly(ADP-ribose) polymerase 1, apoptosis-inducing factor). Severe insults cause accidental cell death that cannot be modulated genetically or by pharmacologic means. However, accidental cell death leads to the release of factors (damage-associated molecular patterns) that initiate systemic effects, as well as inflammation and (regulated) secondary brain injury in neighboring tissue. Furthermore, if one mode of cell death is inhibited, another route may step in at least in a scenario when upstream damaging factors predominate over protective responses. The provision of alternative routes through which the cell undergoes death has to be taken into account in the hunt for novel brain protective strategies.

Mitochondria, Bioenergetics and Excitotoxicity: New Therapeutic Targets in Perinatal Brain Injury.

Injury to the fragile immature brain is implicated in the manifestation of long-term neurological disorders, including childhood disability such as cerebral palsy, learning disability and behavioral disorders. Advancements in perinatal practice and improved care mean the majority of infants suffering from perinatal brain injury will survive, with many subtle clinical symptoms going undiagnosed until later in life. Hypoxic-ischemia is the dominant cause of perinatal brain injury, and constitutes a significant socioeconomic burden to both developed and developing countries. Therapeutic hypothermia is the sole validated clinical intervention to perinatal asphyxia; however it is not always neuroprotective and its utility is limited to developed countries. There is an urgent need to better understand the molecular pathways underlying hypoxic-ischemic injury to identify new therapeutic targets in such a small but critical therapeutic window. Mitochondria are highly implicated following ischemic injury due to their roles as the powerhouse and main energy generators of the cell, as well as cell death processes. While the link between impaired mitochondrial bioenergetics and secondary energy failure following loss of high-energy phosphates is well established after hypoxia-ischemia (HI), there is emerging evidence that the roles of mitochondria in disease extend far beyond this. Indeed, mitochondrial turnover, including processes such as mitochondrial biogenesis, fusion, fission and mitophagy, affect recovery of neurons after injury and mitochondria are involved in the regulation of the innate immune response to inflammation. This review article will explore these mitochondrial pathways, and finally will summarize past and current efforts in targeting these pathways after hypoxic-ischemic injury, as a means of identifying new avenues for clinical intervention.

Human amnion epithelial cells rescue cell death via immunomodulation of microglia in a mouse model of perinatal brain injury.

BACKGROUND: Human amnion epithelial cells (hAECs) are clonogenic and have been proposed to reduce inflammatory-induced tissue injury. Perturbation of the immune response is implicated in the pathogenesis of perinatal brain injury; modulating this response could thus be a novel therapy for treating or preventing such injury. The immunomodulatory properties of hAECs have been shown in other animal models, but a detailed investigation of the effects on brain immune cells following injury has not been undertaken. Here, we investigate the effects of hAECs on microglia, the first immune responders to injury within the brain. METHODS: We generated a mouse model combining neonatal inflammation and perinatal hyperoxia, both of which are risk factors associated with perinatal brain injury. On embryonic day 16 we administered lipopolysaccharide (LPS), or saline (control), intra-amniotically to C57Bl/6 J mouse pups. On postnatal day (P)0, LPS pups were placed in hyperoxia (65% oxygen) and control pups in normoxia for 14 days. Pups were given either hAECs or saline intravenously on P4. RESULTS: At P14, relative to controls, LPS and hyperoxia pups had reduced body weight, increased density of apoptotic cells (TUNEL) in the cortex, striatum and white matter, astrocytes (GFAP) in the white matter and activated microglia (CD68) in the cortex and striatum, but no change in total microglia density (Iba1). hAEC administration rescued the decreased body weight and reduced apoptosis and astrocyte areal coverage in the white matter, but increased the density of total and activated microglia. We then stimulated primary microglia (CD45lowCD11b+) with LPS for 24 h, followed by co-culture with hAEC conditioned medium for 48 h. hAEC conditioned medium increased microglial phagocytic activity, decreased microglia apoptosis and decreased M1 activation markers (CD86). Stimulating hAECs for 24 h with LPS did not alter release of cytokines known to modulate microglia activity. CONCLUSIONS: These data demonstrate that hAECs can directly immunomodulate brain microglia, probably via release of trophic factors. This observation offers promise that hAECs may afford therapeutic utility in the management of perinatal brain injury.

Long-Distance Axonal Growth and Protracted Functional Maturation of Neurons Derived from Human Induced Pluripotent Stem Cells After Intracerebral Transplantation.

The capacity for induced pluripotent stem (iPS) cells to be differentiated into a wide range of neural cell types makes them an attractive donor source for autologous neural transplantation therapies aimed at brain repair. Translation to the in vivo setting has been difficult, however, with mixed results in a wide variety of preclinical models of brain injury and limited information on the basic in vivo properties of neural grafts generated from human iPS cells. Here we have generated a human iPS cell line constitutively expressing green fluorescent protein as a basis to identify and characterize grafts resulting from transplantation of neural progenitors into the adult rat brain. The results show that the grafts contain a mix of neural cell types, at various stages of differentiation, including neurons that establish extensive patterns of axonal growth and progressively develop functional properties over the course of 1 year after implantation. These findings form an important basis for the design and interpretation of preclinical studies using human stem cells for functional circuit re-construction in animal models of brain injury. Stem Cells Translational Medicine 2017;6:1547-1556.

Maternal 25-hydroxyvitamin D is inversely correlated with foetal serotonin.

OBJECTIVE: Maternal vitamin D deficiency during pregnancy has been linked to impaired neurocognitive development in childhood. The mechanism by which vitamin D affects childhood neurocognition is unclear but may be via interactions with serotonin, a neurotransmitter involved in foetal brain development. In this study, we aimed to explore associations between maternal and foetal vitamin D concentrations, and foetal serotonin concentrations at term. STUDY DESIGN AND MEASUREMENTS: Serum 25-hydroxyvitamin D (25(OH)D, nmol/l) and serotonin (5-HT, nmol/l) concentrations were measured in maternal and umbilical cord blood from mother-infant pairs (n = 64). Association between maternal 25(OH)D, cord 25(OH)D and cord serotonin was explored using linear regression, before and after adjusting for maternal serotonin levels. We also assessed the effects of siRNA knockdown of the vitamin D receptor (VDR) and administration of 10 nm 1,25-dihydroxyvitamin D3 on serotonin secretion in human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: We observed an inverse relationship between both maternal and cord 25(OH)D concentrations with cord serotonin concentrations. The treatment of HUVECs with 1,25-dihydroxyvitamin D3 in vitro decreased the release of serotonin (193·9 ±14·8 nmol/l vs 458·9 ± 317·5 nmol/l, control, P < 0·05). Conversely, inactivation of VDR increased serotonin release in cultured HUVECs. CONCLUSIONS: These observations provide the first evidence of an inverse relationship between maternal 25(OH)D and foetal serotonin concentrations. We propose that maternal vitamin D deficiency increases foetal serotonin concentrations and thereby contributes to longer-term neurocognitive impairment in infants and children.

'Neonatal' Nav1.2 reduces neuronal excitability and affects seizure susceptibility and behaviour.

Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels; however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.

Reduced dendritic arborization and hyperexcitability of pyramidal neurons in a Scn1b-based model of Dravet syndrome.

Epileptic encephalopathies, including Dravet syndrome, are severe treatment-resistant epilepsies with developmental regression. We examined a mouse model based on a human ß1 sodium channel subunit (Scn1b) mutation. Homozygous mutant mice shared phenotypic features and pharmaco-sensitivity with Dravet syndrome. Patch-clamp analysis showed that mutant subicular and layer 2/3 pyramidal neurons had increased action potential firing rates, presumably as a consequence of their increased input resistance. These changes were not seen in L5 or CA1 pyramidal neurons. This raised the concept of a regional seizure mechanism that was supported by data showing increased spontaneous synaptic activity in the subiculum but not CA1. Importantly, no changes in firing or synaptic properties of gamma-aminobutyric acidergic interneurons from mutant mice were observed, which is in contrast with Scn1a-based models of Dravet syndrome. Morphological analysis of subicular pyramidal neurons revealed reduced dendritic arborization. The antiepileptic drug retigabine, a K+ channel opener that reduces input resistance, dampened action potential firing and protected mutant mice from thermal seizures. These results suggest a novel mechanism of disease genesis in genetic epilepsy and demonstrate an effective mechanism-based treatment of the disease.

Neurons derived from human embryonic stem cells extend long-distance axonal projections through growth along host white matter tracts after intra-cerebral transplantation.

Human pluripotent stem cells have the capacity for directed differentiation into a wide variety of neuronal subtypes that may be useful for brain repair. While a substantial body of research has lead to a detailed understanding of the ability of neurons in fetal tissue grafts to structurally and functionally integrate after intra-cerebral transplantation, we are only just beginning to understand the in vivo properties of neurons derived from human pluripotent stem cells. Here we have utilized the human embryonic stem (ES) cell line Envy, which constitutively expresses green fluorescent protein (GFP), in order to study the in vivo properties of neurons derived from human ES cells. Rapid and efficient neural induction, followed by differentiation as neurospheres resulted in a GFP+ neural precursor population with traits of neuroepithelial and dorsal forebrain identity. Ten weeks after transplantation into neonatal rats, GFP+ fiber patterns revealed extensive axonal growth in the host brain, particularly along host white matter tracts, although innervation of adjacent nuclei was limited. The grafts were composed of a mix of neural cell types including differentiated neurons and glia, but also dividing neural progenitors and migrating neuroblasts, indicating an incomplete state of maturation at 10 weeks. This was reflected in patch-clamp recordings showing stereotypical properties appropriate for mature functional neurons, including the ability to generate action potentials, as well profiles consistent for more immature neurons. These findings illustrate the intrinsic capacity for neurons derived from human ES cells to integrate at a structural and functional level following transplantation.

Axon initial segment dysfunction in a mouse model of genetic epilepsy with febrile seizures plus.

Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the beta1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type beta1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the beta1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with beta1(C121W) subunits. We therefore conclude that Na+ channel beta1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.