Forest, Wood and Climate Change: Challenges and Opportunities in the UNECE Region

This essay explains the importance of the forests as a factor in addressing the challenges in mitigating climate change. The potential of using the forest sector more fully to capture and store carbon has been limited by the failure of current protocols and other climate change mechanisms to adequately account for the contribution of this sector. Thus, a better accounting, which will give the proper credit to the impacts that this sector is having, is viewed to be an important next step to increasing the resources that countries will devote to this factor in addressing climate change. The degree to which global warming is already affecting the forest is also discussed; increasingly mankind may be required to be more proactive in implementing “planned adaptation” activities such as increasing the diversification of forestry resources.Climate change, global warming, forest, biofuels

Functional regionalization of the differentiating cerebellar Purkinje cell population occurs in an activity-dependent manner

IntroductionThe cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive.Methods and resultsWe show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer.DiscussionHence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves

Dimethylethanolamine Decreases Epileptiform Activity in Acute Human Hippocampal Slices in vitro

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy with about 30% of patients developing pharmacoresistance. These patients continue to suffer from seizures despite polytherapy with antiepileptic drugs (AEDs) and have an increased risk for premature death, thus requiring further efforts for the development of new antiepileptic therapies. The molecule dimethylethanolamine (DMEA) has been tested as a potential treatment in various neurological diseases, albeit the functional mechanism of action was never fully understood. In this study, we investigated the effects of DMEA on neuronal activity in single-cell recordings of primary neuronal cultures. DMEA decreased the frequency of spontaneous synaptic events in a concentration-dependent manner with no apparent effect on resting membrane potential (RMP) or action potential (AP) threshold. We further tested whether DMEA can exert antiepileptic effects in human brain tissue ex vivo. We analyzed the effect of DMEA on epileptiform activity in the CA1 region of the resected hippocampus of TLE patients in vitro by recording extracellular field potentials in the pyramidal cell layer. Epileptiform burst activity in resected hippocampal tissue from TLE patients remained stable over several hours and was pharmacologically suppressed by lacosamide, demonstrating the applicability of our platform to test antiepileptic efficacy. Similar to lacosamide, DMEA also suppressed epileptiform activity in the majority of samples, albeit with variable interindividual effects. In conclusion, DMEA might present a new approach for treatment in pharmacoresistant TLE and further studies will be required to identify its exact mechanism of action and the involved molecular targets

A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH4 as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH4 application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic APOBEC1 dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different APOBEC1 alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the APOBEC1 80I/M-coding alleles as new genetic risk factors for iTLE patients

Cesium activates the neurotransmitter receptor for glycine

The monovalent cations sodium and potassium are crucial for the proper functioning of excitable cells, but, in addition, other monovalent alkali metal ions such as cesium and lithium can also affect neuronal physiology. For instance, there have been recent reports of adverse effects resulting from self-administered high concentrations of cesium in disease conditions, prompting the Food and Drug Administration (FDA) to issue an alert concerning cesium chloride. As we recently found that the monovalent cation NH4+ activates glycine receptors (GlyRs), we investigated the effects of alkali metal ions on the function of the GlyR, which belongs to one of the most widely distributed neurotransmitter receptors in the peripheral and central nervous systems. Whole-cell voltage clamp electrophysiology was performed with HEK293T cells transiently expressing different splice and RNA-edited variants of GlyR α2 and α3 homopentameric channels. By examining the influence of various milli- and sub-millimolar concentrations of lithium, sodium, potassium, and cesium on these GlyRs in comparison to its natural ligand glycine (0.1 mM), we could show that cesium activates GlyRs in a concentration- and post-transcriptional-dependent way. Additionally, we conducted atomistic molecular dynamic simulations on GlyR α3 embedded in a membrane bilayer with potassium and cesium, respectively. The simulations revealed slightly different GlyR-ion binding profiles for potassium and cesium, identifying interactions near the glycine binding pocket (potassium and cesium) and close to the RNA-edited site (cesium) in the extracellular GlyR domain. Together, these findings show that cesium acts as an agonist of GlyRs

Simultaneous impairment of neuronal and metabolic function of mutated gephyrin in a patient with epileptic encephalopathy

Correction to: EMBO Mol Med (2015) 7: 1580–1594. DOI 10.15252/emmm.201505323 | Published online 27 November 2015 EMBO Molecular Medicine 2017 vol 9 No12: 1764.Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition plays an important role in neurological disorders. Gephyrin is a central player at inhibitory postsynapses, directly binds and organizes GABA(A) and glycine receptors (GABA(A)Rs and GlyRs), and is thereby indispensable for normal inhibitory neurotransmission. Additionally, gephyrin catalyzes the synthesis of the molybdenum cofactor (MoCo) in peripheral tissue. We identified a de novo missense mutation (G375D) in the gephyrin gene (GPHN) in a patient with epileptic encephalopathy resembling Dravet syndrome. Although stably expressed and correctly folded, gephyrin-G375D was non-synaptically localized in neurons and acted dominant-negatively on the clustering of wild- type gephyrin leading to a marked decrease in GABA(A)R surface expression and GABAergic signaling. We identified a decreased binding affinity between gephyrin-G375D and the receptors, suggesting that Gly375 is essential for gephyrin-receptor complex formation. Surprisingly, gephyrin-G375D was also unable to synthesize MoCo and activate MoCo-dependent enzymes. Thus, we describe a missense mutation that affects both functions of gephyrin and suggest that the identified defect at GABAergic synapses is the mechanism underlying the patient's severe phenotype.Peer reviewe

Induktion von Synapsen durch Agrin in kultivierten kortikalen Neuronen

The proteoglycan agrin is an integral part and of utmost importance in the development and maintenance of the neuromuscular junction of vertebrates. Even though it is expressed at developmental stages in the central nervous system its role in synaptogenesis is currently unknown. By employing the whole cell patch clamp technique agrin’s effect on the synaptogenesis of cortical neurons in microisland and conventional monolayer cultures was investigated in this thesis. In microisland cultures of single cortical neurons excitatory and inhibitory neurons could be investigated separately. Incubation with soluble agrin for three to five hours led to a robust increase in mEPSC frequency and amplitude in these cultures. Neurotransmitter release and the number of excitatory synapses were investigated. The paired-pulse ratios of agrin- treated cells remained unchanged but the current amplitudes were larger after agrin incubation. Studies on the co-localization of VGlut1+2 and PSD95 showed that treatment with soluble agrin increased the number of co-localized puncta. Stainings with antibodies against AMPAR revealed a larger area of AMPAR clusters and more AMPAR/VGlut1+2 co-localized puncta in agrin-treated cells opposed to VGlut1+2 positive areas. This observation explains the increase in mEPSC amplitude and frequency by an increased number of AMPAR-containing excitatory synapses. Endogenous agrin and the presence of the agrin receptor LRP4 are necessary to induce the increase in mEPSC frequency since LRP4- and agrin-deficient neurons were unable to react to agrin treatment demonstrating the importance of these proteins. The mechanism behind this increase was shown to be related to PI3K/mTOR signaling. Incubation with neuronal agrin led to increased phosphorylation of GSK3β in immunostainings of single cortical neurons and blocking PI3K/mTOR with wortmannin, LY293002 or rapamycin abolished agrin-mediated effects on the increase of the mEPSC frequency. The data suggest that agrin might influence synaptogenesis in cortical neurons by activating PI3K/mTOR signaling leading to the formation of new excitatory synapses. In contrast to microisland cultures conventional monolayer cultures of neurons showed an increase in the frequency of mIPSCs after treatment with soluble agrin for three to five hours. This effect was time and concentration dependent. However changes in the release probability of neurotransmitters and changes in the number of inhibitory synapses were not discovered. Paired-pulse ratios were unaffected by agrin treatment as well as the number of inhibitory synapses. The analysis of agrin-deficient neurons showed no increase in mIPSC frequency after agrin treatment suggesting that endogenous agrin important for proper signal transduction. In summary microisland cultures are a suitable tool to investigate synaptogenesis with electrophysiological and immunocytochemical methods. Using this experimental approach, this study demonstrated that agrin is important during synaptogenesis or synapse maintenance in cortical neurons but not essential.Das Proteoglykan Agrin ist ein integraler Bestandteil von äußerster Wichtigkeit bei der Entwicklung und Aufrechterhaltung der neuromuskulären Endplatte von Vertebraten. Obwohl es auch während der Entwicklung des zentralen Nervensystems exprimiert wird, ist seine Rolle in der Synaptogenese noch ungeklärt. Mit Hilfe der whole-cell Patch-Clamp-Technik wurde die Wirkung von Agrin bei der Synaptogenese von kortikalen Neuronen in microisland und konventionellen Kulturen in dieser Arbeit untersucht. In microisland Kulturen konnten einzelne exzitatorische sowie inhibitorische Neurone getrennt voneinander untersucht werden. Inkubation mit löslichem Agrin führte in diesen Kulturen zu einem robusten Anstieg der mEPSC Frequenz und ihrer Amplitude. Die Neurotransmitterausschüttung und die Anzahl der exzitatorischen Synapsen wurde untersucht. Das Paired-pulse-Verhältnis blieb unverändert verglichen mit unbehandelten Kontrollneuronen aber die Stromamplituden waren größer nach der Inkubation mit Agrin. Kolokalisationsstudien von Vglut1+2 und PSD95 zeigten einen Anstieg der kolokalisierten Puncta nach der Behandlung mit löslichem Agrin. Färbungen mit Antikörpern gerichtet gegen AMPAR offenbarten eine größere Fläche von AMPAR-Anhäufungen gegenüberliegend von VGlut1+2 positiven Arealen sowie mehr kolokalisierte AMPAR/VGlut1+2 puncta in Agrin-behandelten Zellen. Diese Beobachtung erklärt den Anstieg der mEPSC Frequenz und Amplitude durch eine erhöhte Anzahl an AMPAR-enthaltenden exzitatorischen Synapsen. Endogenes Agrin und das Vorhandensein des Agrinrezeptors LRP4 sind notwendig, um den Anstieg der mEPSC-Frequenz zu induzieren, da LRP4- und agrindefiziente Neurone nicht in der Lage waren auf die Agrinbehandlung zu reagieren, was die Wichtigkeit dieser Proteine unterstreicht. Der Mechanismus der hinter diesem Anstieg liegt ist verbunden mit dem PI3K/mTOR Signalweg. Die Inkubation mit löslichem Agrin führte zu einer erhöhten Phosphorylierung von GSK3β in Immunfärbungen von einzelnen kortikalen Neuronen und die Blockade von PI3K/mTOR mit Wortmannin, LY294002 oder Rapamycin hob den Agrin-vermittelten Effekt auf die Zunahme der mEPSC Frequenz auf. Diese Daten lassen vermuten, dass Agrin einen Einfluss auf die Synaptogenese in kortikalen Neuronen durch die Aktivierung des PI3K/mTOR Signalwegs ausübt und zur Bildung neuer exzitatorischer Synapsen führt. Im Unterschied zu microisland Kulturen zeigten konventionelle einschichtige Neuronenkulturen einen Anstieg in der Frequenz von mIPSCs nach einer drei bis fünf stündigen Behandlung mit löslichem Agrin. Dieser Effekt war zeit- und konzentrationsabhängig. Allerdings wurden weder Veränderungen im Verhalten bei der Neurotransmitterausschüttung noch in der Anzahl von inhibitorischen Synapsen entdeckt. Das Paired-pulse-Verhältnis blieb unverändert durch die Agrinbehandlung genau wie die Anzahl der inhibitorischen Synapsen. Die Analyse von agrin-defizienten Neuronen zeigte keinen Anstieg der mIPSC-Frequenz nach Agrinbehandlung was dafür spricht, dass endogenes Agrin wichtig für eine exakte Signaltransduktion ist. Zusammenfassend lässt sich feststellen, dass microisland Kulturen ein geeignetes System darstellen, um Synaptogenese mit elektrophysiologischen und immunocytochemischen Methoden zu untersuchen. In dieser Arbeit konnte mit diesem Versuchsansatz gezeigt werden, dass Agrin die Bildung oder Stabilität von Synapsen in kortikalen Neuronen fördern kann, aber nicht essentiell ist

RNA Editing-Systemic Relevance and Clue to Disease Mechanisms?

Recent advances in sequencing technologies led to the identification of a plethora of different genes and several hundreds of amino acid recoding edited positions. Changes in editing rates of some of these positions were associated with diseases such as atherosclerosis, myopathy, epilepsy, major depression disorder, schizophrenia and other mental disorders as well as cancer and brain tumors. This review article summarizes our current knowledge on that front and presents glycine receptor C-to-U RNA editing as a first example of disease-associated increased RNA editing that includes assessment of disease mechanisms of the corresponding gene product in an animal model

Biallelic gephyrin variants lead to impaired GABAergic inhibition in a patient with developmental and epileptic encephalopathy

Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition is linked to epilepsy. Gephyrin (Geph) is the principal scaffolding protein at inhibitory synapses and is essential for postsynaptic clustering of glycine (GlyRs) and GABA type A receptors. Consequently, gephyrin is crucial for maintaining the relationship between excitation and inhibition in normal brain function and mutations in the gephyrin gene (GPHN) are associated with neurodevelopmental disorders and epilepsy. We identified bi-allelic variants in the GPHN gene, namely the missense mutation c.1264G > A and splice acceptor variant c.1315-2A > G, in a patient with developmental and epileptic encephalopathy. We demonstrate that the splice acceptor variant leads to nonsense-mediated mRNA decay. Furthermore, the missense variant (D422N) alters gephyrin structure, as examined by analytical size exclusion chromatography and circular dichroism-spectroscopy, thus leading to reduced receptor clustering and sensitivity towards calpain-mediated cleavage. In addition, both alterations contribute to an observed reduction of inhibitory signal transmission in neurons, which likely contributes to the pathological encephalopathy