Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity.

Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.

A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels.

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies.

Maternal alcohol consumption in pregnancy enhances arterial stiffness and alters vasodilator function that varies between vascular beds in fetal sheep.

While the impact of alcohol consumption by pregnant women on fetal neurodevelopment has received much attention, the effects on the cardiovascular system are not well understood. We hypothesised that repeated exposure to alcohol (ethanol) in utero would alter fetal arterial reactivity and wall stiffness, key mechanisms leading to cardiovascular disease in adulthood. Ethanol (0.75 g (kg body weight)(-1)) was infused intravenously into ewes over 1 h daily for 39 days in late pregnancy (days 95-133 of pregnancy, term ∼147 days). Maternal and fetal plasma ethanol concentrations at the end of the hour were ∼115 mg dl(-1), and then declined to apparent zero over 8 h. At necropsy (day 134), fetal body weight and fetal brain-body weight ratio were not affected by alcohol infusion. Small arteries (250-300 µm outside diameter) from coronary, renal, mesenteric, femoral (psoas) and cerebral beds were isolated. Endothelium-dependent vasodilatation sensitivity was reduced 10-fold in coronary resistance arteries, associated with a reduction in endothelial nitric oxide synthase mRNA (P = 0.008). Conversely, vasodilatation sensitivity was enhanced 10-fold in mesenteric and renal resistance arteries. Arterial stiffness was markedly increased (P = 0.0001) in all five vascular beds associated with an increase in elastic modulus and, in cerebral vessels, with an increase in collagen Iα mRNA. Thus, we show for the first time that fetal arteries undergo marked and regionally variable adaptations as a consequence of repeated alcohol exposure. These alcohol-induced vascular effects occurred in the apparent absence of fetal physical abnormalities or fetal growth restriction.

Maternal melatonin administration mitigates coronary stiffness and endothelial dysfunction, and improves heart resilience to insult in growth restricted lambs.

Intrauterine growth restriction (IUGR) is associated with impaired cardiac function in childhood and is linked to short- and long-term morbidities. Placental dysfunction underlies most IUGR, and causes fetal oxidative stress which may impact on cardiac development. Accordingly, we investigated whether antenatal melatonin treatment, which possesses antioxidant properties, may afford cardiovascular protection in these vulnerable fetuses. IUGR was induced in sheep fetuses using single umbilical artery ligation on day 105-110 of pregnancy (term 147). Study 1: melatonin (2 mg h(-1)) was administered i.v. to ewes on days 5 and 6 after surgery. On day 7 fetal heart function was assessed using a Langendorff apparatus. Study 2: a lower dose of melatonin (0.25 mg h(-1)) was administered continuously following IUGR induction and the ewes gave birth normally at term. Lambs were killed when 24 h old and coronary vessels studied. Melatonin significantly improved fetal oxygenation in vivo. Contractile function in the right ventricle and coronary flow were enhanced by melatonin. Ischaemia-reperfusion-induced infarct area was 3-fold greater in IUGR hearts than in controls and this increase was prevented by melatonin. In isolated neonatal coronary arteries, endothelium-dependent nitric oxide (NO) bioavailability was reduced in IUGR, and was rescued by modest melatonin treatment. Melatonin exposure also induced the emergence of an indomethacin-sensitive vasodilation. IUGR caused marked stiffening of the coronary artery and this was prevented by melatonin. Maternal melatonin treatment reduces fetal hypoxaemia, improves heart function and coronary blood flow and rescues cardio-coronary deficit induced by IUGR.

Over-expression of RCAN1 causes Down syndrome-like hippocampal deficits that alter learning and memory.

People with Down syndrome (DS) exhibit abnormal brain structure. Alterations affecting neurotransmission and signalling pathways that govern brain function are also evident. A large number of genes are simultaneously expressed at abnormal levels in DS; therefore, it is a challenge to determine which gene(s) contribute to specific abnormalities, and then identify the key molecular pathways involved. We generated RCAN1-TG mice to study the consequences of RCAN1 over-expression and investigate the contribution of RCAN1 to the brain phenotype of DS. RCAN1-TG mice exhibit structural brain abnormalities in those areas affected in DS. The volume and number of neurons within the hippocampus is reduced and this correlates with a defect in adult neurogenesis. The density of dendritic spines on RCAN1-TG hippocampal pyramidal neurons is also reduced. Deficits in hippocampal-dependent learning and short- and long-term memory are accompanied by a failure to maintain long-term potentiation (LTP) in hippocampal slices. In response to LTP induction, we observed diminished calcium transients and decreased phosphorylation of CaMKII and ERK1/2-proteins that are essential for the maintenance of LTP and formation of memory. Our data strongly suggest that RCAN1 plays an important role in normal brain development and function and its up-regulation likely contributes to the neural deficits associated with DS.

Towards understanding sex differences in autism spectrum disorders.

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by social deficits, repetitive behaviours and lack of empathy. Its significant genetic heritability and potential comorbidities often lead to diagnostic and therapeutic challenges. This review addresses the biological basis of ASD, focusing on the sex differences in gene expression and hormonal influences. ASD is more commonly diagnosed in males at a ratio of 4:1, indicating a potential oversight in female-specific ASD research and a risk of underdiagnosis in females. We consider how ASD manifests differently across sexes by exploring differential gene expression in female and male brains and consider how variations in steroid hormones influence ASD characteristics. Synaptic function, including excitation/inhibition ratio imbalance, is influenced by gene mutations and this is explored as a key factor in the cognitive and behavioural manifestations of ASD. We also discuss the role of micro RNAs (miRNAs) and highlight a novel mutation in miRNA-873, which affects a suite of key synaptic genes, neurexin, neuroligin, SHANK and post-synaptic density proteins, implicated in the pathology of ASD. Our review suggests that genetic predisposition, sex differences in brain gene expression, and hormonal factors significantly contribute to the presentation, identification and severity of ASD, necessitating sex-specific considerations in diagnosis and treatments. These findings advocate for personalized interventions to improve the outcomes for individuals with ASD.

3D Functional Neuronal Networks in Free-Standing Bioprinted Hydrogel Constructs.

The composition, elasticity, and organization of the extracellular matrix within the central nervous system contribute to the architecture and function of the brain. From an in vitro modeling perspective, soft biomaterials are needed to mimic the 3D neural microenvironments. While many studies have investigated 3D culture and neural network formation in bulk hydrogel systems, these approaches have limited ability to position cells to mimic sophisticated brain architectures. In this study, cortical neurons and astrocytes acutely isolated from the brains of rats are bioprinted in a hydrogel to form 3D neuronal constructs. Successful bioprinting of cellular and acellular strands in a multi-bioink approach allows the subsequent formation of gray- and white-matter tracts reminiscent of cortical structures. Immunohistochemistry shows the formation of dense, 3D axon networks. Calcium signaling and extracellular electrophysiology in these 3D neuronal networks confirm spontaneous activity in addition to evoked activities under pharmacological and electrical stimulation. The system and bioprinting approaches are capable of fabricating soft, free-standing neuronal structures of different bioink and cell types with high resolution and throughput, which provide a promising platform for understanding fundamental questions of neural networks, engineering neuromorphic circuits, and for in vitro drug screening.

Rapid electrophoretic deposition of biocompatible graphene coatings for high-performance recording neural electrodes.

The electrical and biological interfacial properties of invasive electrodes have a significant impact on the performance and longevity of neural recordings in the brain. In this study, we demonstrated rapid electrophoretic deposition and electrochemical reduction of graphene oxide (GO) on metal-based neural electrodes. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and other characterizations confirmed the existence of a uniform and effectively reduced graphene oxide coating. Electrochemically reduced graphene oxide (ErGO) coated Pt/Ir neural electrodes exhibited 15.2-fold increase in charge storage capacity (CSC) and 90% decrease in impedance with only 3.8% increase in electrode diameter. Patch clamp electrophysiology and calcium imaging of primary rat hippocampus neurons cultured on ErGO demonstrated that there was no adverse impact on the functional development of neurons. Immunostaining showed a balanced growth of excitatory and inhibitory neurons, and astrocytes. Acute recordings from the auditory cortex and chronic recordings (19 days) from the somatosensory cortex found ErGO coating improved the performance of neural electrodes in signal-to-noise ratio (SNR) and amplitude of signals. The proposed approach not only provides an in-depth evaluation of the effect of ErGO coating on neural electrodes but also widens the coating methods of commercial neural electrodes.

Vitamin D insufficiency is associated with impaired vascular endothelial and smooth muscle function and hypertension in young rats.

Increasing evidence links vitamin D deficiency and cardiovascular dysfunction in human adults. There is a worldwide increase in the prevalence of vitamin D deficiency in women of reproductive age, particularly dark-skinned and/or veiled women and their infants. We used a rat model to determine the functional impact of vitamin D deficiency during intra uterine and early life on resistance artery reactivity and blood pressure in the offspring as young adults. Rat dams were maintained on vitamin D deficient or replete chow before and during pregnancy and lactation. The offspring were maintained on the same chow until studied at 7-8 weeks of age. Conscious blood pressure was measured. Endothelial and smooth muscle function were tested in mesenteric arteries on a pressure myograph. Vitamin D deficient male and female offspring had a 10-fold lower serum 25-hydroxyvitamin D (P < 0.0001) and markedly elevated blood pressures (11-20 mmHg, P < 0.001) and heart rates (21-40 beats min(-1), P < 0.02) than control fed offspring. Serum calcium was unchanged. Mesenteric artery myogenic tone was doubled in vitamin D deficiency. Endothelium-derived nitric oxide-evoked dilation was halved in arteries from vitamin D deficient males and dioestrous females. Dilation attributed to endothelium-derived hyperpolarizing factor was all but abolished in vitamin D deficient oestrous females. Nitroprusside-evoked dilation was unaltered in arteries from males, but was markedly reduced in vessels of vitamin D deplete females. In conclusion, early life vitamin D deficiency is associated with endothelial vasodilator dysfunction, and this is likely to contribute to the accompanying elevation in blood pressure and an increased cardiovascular disease risk.

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells.

Gram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gram-negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram-negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kappaB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice-induced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

Esterase-Mediated Sustained Release of Peptide-Based Therapeutics from a Self-Assembled Injectable Hydrogel.

A synthetic strategy for conjugating small molecules and peptide-based therapeutics, via a cleavable ester bond, to a lipidated ß3-tripeptide is presented. The drug-loaded ß3-peptide was successfully co-assembled with a functionally inert lipidated ß3-tripeptide to form a hydrogel. Quantitative release of lactose from the hydrogel, by the action of serum esterases, is demonstrated over 28 days. The esterase-mediated sustained release of the bioactive brain-derived neurotrophic factor (BDNF) peptide mimics from the hydrogel resulted in increased neuronal survival and normal neuronal function of peripheral neurons. These studies define a versatile strategy for the facile synthesis and co-assembly of self-assembling ß3-peptide-based hydrogels with the ability to control drug release using endogenous esterases with potential in vivo applications for sustained localized drug delivery.

Prenatal alcohol exposure: implications for cardiovascular function in the fetus and beyond.

1. The effects of heavy maternal alcohol consumption during pregnancy on cognitive and behavioural performance and craniofacial malformations in the offspring have been studied extensively. In contrast, the impact of maternal alcohol intake on the cardiovascular system of the offspring and the effects of more modest consumption have received very scant consideration. 2. Adverse conditions in the pre- and neonatal periods can have a profound legacy on offspring health, including the risk of cardiovascular disease. Prenatal alcohol exposure can modulate vascular reactivity, including endothelial and smooth muscle function. 3. Other effects of prenatal alcohol exposure are emerging, including impairment of nephrogenesis and kidney function and increased arterial stiffness. The impact of even modest prenatal alcohol exposure on cardiovascular health in the offspring remains to be determined. 4. It is envisaged that the culmination of reduced renal and vascular capacity will render the offspring more vulnerable to cardiovascular disease with ageing and exposure to additional insults and lifestyle factors.

Autism-associated miR-873 regulates ARID1B, SHANK3 and NRXN2 involved in neurodevelopment.

Autism spectrum disorders (ASD) are highly heritable neurodevelopmental disorders with significant genetic heterogeneity. Noncoding microRNAs (miRNAs) are recognised as playing key roles in development of ASD albeit the function of these regulatory genes remains unclear. We previously conducted whole-exome sequencing of Australian families with ASD and identified four novel single nucleotide variations in mature miRNA sequences. A pull-down transcriptome analysis using transfected SH-SY5Y cells proposed a mechanistic model to examine changes in binding affinity associated with a unique mutation found in the conserved 'seed' region of miR-873-5p (rs777143952: T > A). Results suggested several ASD-risk genes were differentially targeted by wild-type and mutant miR-873 variants. In the current study, a dual-luciferase reporter assay confirmed miR-873 variants have a 20-30% inhibition/dysregulation effect on candidate autism risk genes ARID1B, SHANK3 and NRXN2 and also confirmed the affected expression with qPCR. In vitro mouse hippocampal neurons transfected with mutant miR-873 showed less morphological complexity and enhanced sodium currents and excitatory neurotransmission compared to cells transfected with wild-type miR-873. A second in vitro study showed CRISPR/Cas9 miR-873 disrupted SH-SY5Y neuroblastoma cells acquired a neuronal-like morphology and increased expression of ASD important genes ARID1B, SHANK3, ADNP2, ANK2 and CHD8. These results represent the first functional evidence that miR-873 regulates key neural genes involved in development and cell differentiation.

Null mutation of the alpha4 nicotinic receptor subunit increases the propensity of muscarinic-mediated neuronal bursting in mouse hippocampal slices.

Alpha4 subunit nicotinic cholinergic receptor (nAChR) knock out mice (KO) have a greater susceptibility to proconvulsant-induced seizures than do wild type (WT). The underlying mechanisms remain obscure. We tested whether such seizure-like activity was reflected in bursting activity of hippocampal neurons by recording with intracellular microelectrodes from CA1 pyramidal neurons in slices from WT and KO mice. Intriguingly, while carbachol-induced bursting activity occurred in only 21% of WT slices, qualitatively identical patterns of bursting occurred in 72% of KO slices. Extracellular recordings from CA1 and CA3 regions suggest that carbachol-mediated population activity was regionalized in our preparations. The relative weighting of excitatory to inhibitory synaptic potentials was similar between WT and alpha4 KO mice. However, burst-firing cells had a smaller input time constant than non-bursters. Low-concentration DHbetaE (selective alpha4beta2 nAChR antagonist) did not increase the propensity of WT slices to burst-fire, indicating that absence of alpha4 subunits per se, cannot explain the differences in activity between slices from WT and KO mice. These observations suggest that alpha4 nAChRs are unlikely to be involved in modulating the pattern of bursting neural activity, but their absence could induce subtle developmental changes in the sensitivity of hippocampal circuits to develop this behaviour.

Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor.

The nature of the vasodilator endothelium-derived hyperpolarizing factor (EDHF) is controversial, putatively involving diffusible factors and/or electrotonic spread of hyperpolarization generated in the endothelium via myoendothelial gap junctions (MEGJs). In this study, we investigated the relationship between the existence of MEGJs, endothelial cell (EC) hyperpolarization, and EDHF-attributed smooth muscle cell (SMC) hyperpolarization in two different arteries: the rat mesenteric artery, where EDHF-mediated vasodilation is prominent, and the femoral artery, where there is no EDHF-dependent relaxation. In the rat mesenteric artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization of both ECs and SMCs, and characteristic pentalaminar MEGJs were found connecting the two cell layers. In contrast, in the femoral artery, ACh evoked hyperpolarization in only ECs but not in SMCs, and no MEGJs were present. Selective hyperpolarization of ECs or SMCs evoked hyperpolarization in the other cell type in the mesenteric artery but not in the femoral artery. Disruption of gap junctional coupling using the peptide Gap 27 markedly reduced the ACh-induced hyperpolarization in SMCs, but not in ECs, of the mesenteric artery. These results show that transfer of EC hyperpolarization or of a small molecule to SMCs through MEGJs is essential and sufficient to explain EDHF.

Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain.

Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration.

Learning, memory and long-term potentiation are altered in Nedd4 heterozygous mice.

The consolidation of short-term memory into long-term memory involves changing protein level and activity for the synaptic plasticity required for long-term potentiation (LTP). AMPA receptor trafficking is a key determinant of LTP and recently ubiquitination by Nedd4 has been shown to play an important role via direct action on the GluA1 subunit, although the physiological relevance of these findings are yet to be determined. We therefore investigated learning and memory in Nedd4(+/-) mice that have a 50% reduction in levels of Nedd4. These mice showed decreased long-term spatial memory as evidenced by significant increases in the time taken to learn the location of and subsequently find a platform in the Morris water maze. In contrast, there were no significant differences between Nedd4(+/+) and Nedd4(+/-) mice in terms of short-term spatial memory in a Y-maze test. Nedd4(+/-) mice also displayed a significant reduction in post-synaptic LTP measured in hippocampal brain slices. Immunofluorescence of Nedd4 in the hippocampus confirmed its expression in hippocampal neurons of the CA1 region. These findings indicate that reducing Nedd4 protein by 50% significantly impairs LTP and long-term memory thereby demonstrating an important role for Nedd4 in these processes.