Dirk Isbrandt

Adenosine Receptor Antagonists Including Caffeine Alter Fetal Brain Development in Mice

Exposure to adenosine receptor antagonists in utero affects fetal brain development in mice. Adenosine Receptor Antagonists and Fetal Brain Development Neural development is strongly influenced by environmental factors including certain drugs. Little information is available about the effects of adenosine receptor antagonists such as caffeine on neural development. To address this question, Silva et al. added caffeine to the drinking water of female mice throughout pregnancy and lactation. They found that caffeine or an adenosine receptor antagonist that specifically blocks type 2A adenosine receptors delayed the migration of specific populations of neurons during brain maturation, resulting in their delayed insertion into target regions. They then showed that 1-week-old offspring of pregnant mice treated with adenosine receptor antagonists were more susceptible to seizures when exposed to a seizure-inducing agent. They further demonstrated that adult offspring of pregnant mice treated with adenosine receptor antagonists had reduced numbers of certain neuronal types as well as impaired memory on certain types of memory tests. This study raises questions about the effects of adenosine receptor antagonists including caffeine on brain development in humans. Retrospective and longitudinal prospective human studies will be needed to evaluate the consequences of caffeine consumption during pregnancy. Consumption of certain substances during pregnancy can interfere with brain development, leading to deleterious long-term neurological and cognitive impairments in offspring. To test whether modulators of adenosine receptors affect neural development, we exposed mouse dams to a subtype-selective adenosine type 2A receptor (A2AR) antagonist or to caffeine, a naturally occurring adenosine receptor antagonist, during pregnancy and lactation. We observed delayed migration and insertion of γ-aminobutyric acid (GABA) neurons into the hippocampal circuitry during the first postnatal week in offspring of dams treated with the A2AR antagonist or caffeine. This was associated with increased neuronal network excitability and increased susceptibility to seizures in response to a seizure-inducing agent. Adult offspring of mouse dams exposed to A2AR antagonists during pregnancy and lactation displayed loss of hippocampal GABA neurons and some cognitive deficits. These results demonstrate that exposure to A2AR antagonists including caffeine during pregnancy and lactation in rodents may have adverse effects on the neural development of their offspring.

Homoarginine supplementation improves blood glucose in diet-induced obese mice.

Abstractl-Homoarginine (hArg) is an endogenous amino acid which has emerged as a novel biomarker for stroke and cardiovascular disease. Low circulating hArg levels are associated with increased mortality and vascular events, whereas recent data have revealed positive correlations between circulating hArg and metabolic vascular risk factors like obesity or blood glucose levels. However, it is unclear whether hArg levels are causally linked to metabolic parameters. Therefore, the aim of our study was to investigate whether hArg directly influences body weight, blood glucose, glucose tolerance or insulin sensitivity. Here, we show that hArg supplementation (14 and 28xa0mg/mL orally per drinking water) ameliorates blood glucose levels in mice on high-fat diet (HFD) by a reduction of 7.3xa0±xa03.7 or 13.4xa0±xa03.8xa0%, respectively. Fasting insulin concentrations were slightly, yet significantly affected (63.8xa0±xa011.3 or 162.1xa0±xa039.5xa0% of control animals, respectively), whereas body weight and glucose tolerance were unaltered. The substantial augmentation of hArg plasma concentrations in supplemented animals (327.5xa0±xa040.4 or 627.5xa0±xa060.3xa0% of control animals, respectively) diminished profoundly after the animals became obese (129.9xa0±xa016.6xa0% in control animals after HFD vs. 140.1xa0±xa08.5 or 206.3xa0±xa013.6xa0%, respectively). This hArg-lowering effect may contribute to the discrepancy between the inverse correlation of plasma hArg levels with stroke and cardiovascular outcome, on thexa0one hand, and the direct correlation with cardiovascular risk factors like obesity and blood glucose, on the other hand, that has been observed in human studies. Our results suggest that the glucose-lowering effects of hArg may reflect a compensatory mechanism of blood glucose reduction by hArg upregulation in obese individuals, without directly influencing body weight or glucose tolerance.

Activity of NaV1.2 promotes neurodegeneration in an animal model of multiple sclerosis

Counteracting the progressive neurological disability caused by neuronal and axonal loss is the major unmet clinical need in multiple sclerosis therapy. However, the mechanisms underlying irreversible neuroaxonal degeneration in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are not well understood. A long-standing hypothesis holds that the distribution of voltage-gated sodium channels along demyelinated axons contributes to neurodegeneration by increasing neuroaxonal sodium influx and energy demand during CNS inflammation. Here, we tested this hypothesis in vivo by inserting a human gain-of-function mutation in the mouse NaV1.2-encoding gene Scn2a that is known to increase NaV1.2-mediated persistent sodium currents. In mutant mice, CNS inflammation during EAE leads to elevated neuroaxonal degeneration and increased disability and lethality compared with wild-type littermate controls. Importantly, immune cell infiltrates were not different between mutant EAE mice and wild-type EAE mice. Thus, this study shows that increased neuronal NaV1.2 activity exacerbates inflammation-induced neurodegeneration irrespective of immune cell alterations and identifies NaV1.2 as a promising neuroprotective drug target in multiple sclerosis.

Transcriptomic and metabolic analyses reveal salvage pathways in creatine-deficient AGAT−/− mice

Skeletal muscles require energy either at constant low (e.g., standing and posture) or immediate high rates (e.g., exercise). To fulfill these requirements, myocytes utilize the phosphocreatine (PCr)/creatine (Cr) system as a fast energy buffer and shuttle. We have generated mice lacking l-arginine:glycine amidino transferase (AGAT), the first enzyme of creatine biosynthesis. These AGAT−/− (d/d) mice are devoid of the PCr/Cr system and reveal severely altered oxidative phosphorylation. In addition, they exhibit complete resistance to diet-induced obesity, which is associated with a chronic activation of AMP-activated protein kinase in muscle and white adipose tissue. The underlying metabolic rearrangements have not yet been further analyzed. Here, we performed gene expression analysis in skeletal muscle and a serum amino acid profile of d/d mice revealing transcriptomic and metabolic alterations in pyruvate and glucose pathways. Differential pyruvate tolerance tests demonstrated preferential conversion of pyruvate to alanine, which was supported by increased protein levels of enzymes involved in pyruvate and alanine metabolism. Pyruvate tolerance tests suggested severely impaired hepatic gluconeogenesis despite increased availability of pyruvate and alanine. Furthermore, enzymes of serine production and one-carbon metabolism were significantly up-regulated in d/d mice, indicating increased de novo formation of one-carbon units from carbohydrate metabolism linked to NAD(P)H production. Besides the well-established function of the PCr/Cr system in energy metabolism, our transcriptomic and metabolic analyses suggest that it plays a pivotal role in systemic one-carbon metabolism, oxidation/reduction, and biosynthetic processes. Therefore, the PCr/Cr system is not only an energy buffer and shuttle, but also a crucial component involved in numerous systemic metabolic processes.

Early-life exposure to caffeine affects the construction and activity of cortical networks in mice

Abstract The consumption of psychoactive drugs during pregnancy can have deleterious effects on newborns. It remains unclear whether early‐life exposure to caffeine, the most widely consumed psychoactive substance, alters brain development. We hypothesized that maternal caffeine ingestion during pregnancy and the early postnatal period in mice affects the construction and activity of cortical networks in offspring. To test this hypothesis, we focused on primary visual cortex (V1) as a model neocortical region. In a study design mimicking the daily consumption of approximately three cups of coffee during pregnancy in humans, caffeine was added to the drinking water of female mice and their offspring were compared to control offspring. Caffeine altered the construction of GABAergic neuronal networks in V1, as reflected by a reduced number of somatostatin‐containing GABA neurons at postnatal days 6–7, with the remaining ones showing poorly developed dendritic arbors. These findings were accompanied by increased synaptic activity in vitro and elevated network activity in vivo in V1. Similarly, in vivo hippocampal network activity was altered from the neonatal period until adulthood. Finally, caffeine‐exposed offspring showed increased seizure susceptibility in a hyperthermia‐induced seizure model. In summary, our results indicate detrimental effects of developmental caffeine exposure on mouse brain development. HighlightsIn mice, early‐life caffeine exposure disrupted development of cortical networks.Caffeine exposure transiently reduced number of interneurons in V1 cortex.In vitro and in vivo neuronal activity in V1 were increased after caffeine exposure.Caffeine exposure persistently increased hippocampal network activity in vivo.Susceptibility to febrile seizures was increased in caffeine exposed pups.

GABAergic Transmission during Brain Development: Multiple Effects at Multiple Stages:

In recent years, considerable progress has been achieved in deciphering the cellular and network functions of GABAergic transmission in the intact developing brain. First, in vivo studies in non-mammalian and mammalian species confirmed the long-held assumption that GABA acts as a mainly depolarizing neurotransmitter at early developmental stages. At the same time, GABAergic transmission was shown to spatiotemporally constrain spontaneous cortical activity, whereas firm evidence for GABAergic excitation in vivo is currently missing. Second, there is a growing body of evidence indicating that depolarizing GABA may contribute to the activity-dependent refinement of neural circuits. Third, alterations in GABA actions have been causally linked to developmental brain disorders and identified as potential targets of timed prophylactic interventions. In this article, we review these major recent findings and argue that both depolarizing and inhibitory GABA actions may be crucial for physiological brain maturation.

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

Impaired cardiac contractile function in arginine:glycine amidinotransferase knockout mice devoid of creatine is rescued by homoarginine but not creatine

Abstract Aims Creatine buffers cellular adenosine triphosphate (ATP) via the creatine kinase reaction. Creatine levels are reduced in heart failure, but their contribution to pathophysiology is unclear. Arginine:glycine amidinotransferase (AGAT) in the kidney catalyses both the first step in creatine biosynthesis as well as homoarginine (HA) synthesis. AGAT-/- mice fed a creatine-free diet have a whole body creatine-deficiency. We hypothesized that AGAT-/- mice would develop cardiac dysfunction and rescue by dietary creatine would imply causality. Methods and results Withdrawal of dietary creatine in AGAT-/- mice provided an estimate of myocardial creatine efflux of ∼2.7%/day; however, in vivo cardiac function was maintained despite low levels of myocardial creatine. Using AGAT-/- mice naïve to dietary creatine we confirmed absence of phosphocreatine in the heart, but crucially, ATP levels were unchanged. Potential compensatory adaptations were absent, AMPK was not activated and respiration in isolated mitochondria was normal. AGAT-/- mice had rescuable changes in body water and organ weights suggesting a role for creatine as a compatible osmolyte. Creatine-naïve AGAT-/- mice had haemodynamic impairment with low LV systolic pressure and reduced inotropy, lusitropy, and contractile reserve. Creatine supplementation only corrected systolic pressure despite normalization of myocardial creatine. AGAT-/- mice had low plasma HA and supplementation completely rescued all other haemodynamic parameters. Contractile dysfunction in AGAT-/- was confirmed in Langendorff perfused hearts and in creatine-replete isolated cardiomyocytes, indicating that HA is necessary for normal cardiac function. Conclusions Our findings argue against low myocardial creatine per se as a major contributor to cardiac dysfunction. Conversely, we show that HA deficiency can impair cardiac function, which may explain why low HA is an independent risk factor for multiple cardiovascular diseases.

A mechanistic link between glia and neuronal excitability in acute neuroinflammation

Activation of the innate immune system can induce inflammatory responses in the brain causing transient or persistent changes in brain structure, cognition, and behaviour. Experimental and clinical research of the last decade has revealed inflammatory mediators that act on the brain, which are produced either in the periphery as a consequence of systemic infection, or in the brain following seizures, neurotrauma, stroke, or neurodegenerative processes. Brain inflammation, in turn, may alter neuronal excitability and promote the genesis of seizures, thus potentially contributing to a self-potentiating cycle of glial and neuronal activation (Vezzani et al. 2011). A widely used experimental approach to studying brain inflammation is the peripheral or intracranial application of lipopolysaccharide (LPS) endotoxin predominantly activating the Toll-like receptor (TLR) signalling pathway via binding to microglial TLR4, which is upregulated following brain inflammation and considered to be the primary receptor mediating LPS-induced microglial activation. As overactive microglia can induce neurotoxic effects by the excess production of cytotoxic factors such as superoxide, nitric oxide (NO) and tumour necrosis factor-α (TNFα), microglia can cause additional neuronal loss or increase ongoing neuronal damage. Only a few of the mechanisms leading to neuronal and network hyperexcitability as a direct effect of acute inflammation are known, which include cytokine-induced effects on synaptic transmission and plasticity, and changes in intrinsic properties determined by ion channel composition or function (Vezzani & Viviani, 2015). In this issue of The Journal of Physiology, Tzour et al. (2017) present a novel mechanism with the potential to facilitate translational therapies. They demonstrate that acute LPS-induced neuronal hyperexcitability in rat acute hippocampal slices is mediated by a complex glia-to-neuron signalling cascade that finally results in a functional downregulation of KV7 voltage-gated potassium channels, also known as M-channels. Using a combination of single-cell electrophysiology, pharmacology and calcium imaging, they established a signalling cascade induced by acute LPS application with sequential activation of microglia, astrocytes and neurons leading to a functional loss of KV7/M-channel activity in CA1 pyramidal neurons. Neuronal KV7 channels, which are widely expressed in the brain, are mainly localized to presynaptic compartments such as axon initial segments and axons. As they activate close to the resting membrane potential and control subthreshold excitability and spike generation, KV7/M-channels are important regulators of neuronal excitability (Delmas & Brown, 2005). This role is also suggested by the genetic link between human epilepsy syndromes and KV7 subunit dysfunction caused by mutations in KCNQ2 and KCNQ3, which encode the KV7.2 and KV7.3 α-subunits of M-channels. KV7/M-channel activity is modulated by neurotransmitters and hormones, including acetylcholine, glutamate and many others, through activation of G protein-coupled receptors leading to their inhibition (Delmas & Brown, 2005). Starting from the finding that acute LPS application in murine brain slices triggers an increase in neuronal excitability of hippocampal neurons via microglia and astrocytes (Pascual et al. 2012), Tzour and colleagues found that it was the activation of neuronal group I metabotropic glutamate receptors (specifically mGluR5) through glutamate released from astrocytes that caused the release of Ca2+ from internal stores, the subsequent inhibition of KV7/M-currents and, thereby, the concomitant increase in neuronal excitability. For LPS to exert this action, activation of purinergic receptors on astrocytes by ATP – which is likely to originate from microglia – was required. Notably, application of the novel anticonvulsant drug retigabine, a KV7 channel opener that acts by shifting the voltage dependence to more hyperpolarized membrane potentials, reversed the excitatory LPS effects. If the attenuation of KV7/M currents turns out to be a general finding in neuroinflammation-induced hyperexcitability, drugs enhancing M-channels could be a viable therapeutic option stabilizing cellular and network excitability. In this context, it is of note that retigabine is neuroprotective in rodent models of ischaemic stroke when administered within a critical period after the insult (Bierbower et al. 2015). These neuroprotective effects can be attributed to the prevention of ischaemia-induced neuronal hyperexcitability following a massive release of glutamate induced by cell injury or neuronal dysfunction. In addition, retigabine also attenuated the inflammatory response by limiting the expression of CD40L, a membrane protein of the TNF receptor family playing an important role in the inflammatory response after acute ischaemic infarction (Bierbower et al. 2015). It remains to be investigated, however, whether the attenuation of KV7/M-currents also plays a role in chronic neuroinflammation, because homeostatic processes such as activity-dependent transcriptional upregulation of KV7 subunits (Zhang & Shapiro, 2012), or augmentation by reactive oxygen species (ROS), may preserve M-channel function. As KV7 channels are attractive pharmacological targets and retigabine has already been used as an anticonvulsant in human epilepsy patients, the present data encourage further studies addressing the potential benefit of enhancing KV7 currents in neuroinflammation.

Small area, low power neural recording integrated circuit in 130 nm CMOS technology for small mammalians

In neuroscience research the development of the brain and the treatment of diseases like certain forms of epilepsy is analysed with genetic mouse disease models. For the special case of the recording from neonatal mice a custom designed integrated circuit is presented. Neonatal mice are only two to three centimetres large and have a weight of only a few gram. Thus, the recording circuitry has to be very small and light weight. The integrated circuit implements 16 low-area, low-power analogue differential preamplifiers with a bandpass characteristic (0.5 Hz to 10 kHz). A multiplexed structure of 8:1 multiplexer, post amplifier and 10 bit successive approximation register (SAR) analogue-to-digital converter (ADC) digitizes the signals with high resolution. The digital data is transmitted via a Serial Peripheral Interface (SPI). The integrated circuit has been implemented in a 130 nm CMOS technology and has been successfully applied in in-vivo measurements with an adult mouse.