Genetic Techniques and Circuit Analysis

Reaching an understanding of how neuronal circuits work and what they compute is a fundamental aim of neuroscience, perhaps even the most fundamental. We have to both establish the connections between cell types and reversibly manipulate their activity cell-typeselectively. Such work sounds in principle straight-forward, but it has been difficult to achieve. This has now all changed. There has been a quite remarkable development of genetic techniques published in the last years, so that the topic of “Genetic techniques and circuit analysis ” covered by the articles in this Special Issue is truly flourishing. The extremely easy applicability of the channelrhodopsin-2 system (ChR2) in diverse animals and circuit settings has been a phenomenal breakthrough and captured the imagination of the neuroscience community (see, for example, Adamantidis et al., 2009; Han et al., 2009). A major advantage of ChR2 is that precise patterns of activation can be delivered cell-type selectively

E-I Balance and Human Diseases – from Molecules to Networking

Information transfer in the brain requires a homeostatic control of neuronal excitability. Therefore, a functional balance between excitatory and inhibitory systems is established during development. This review contains recent information about the molecular mechanisms orchestrating the establishment and maintenance of this excitation-inhibition (E-I) balance, and it reviews examples of deregulation of inhibitory and excitatory systems at a molecular, network and disease level of investigation

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

Phorbol-Ester Mediated Suppression of hASH1 Synthesis: Multiple Ways to Keep the Level Down

Human achaete-scute homolog-1 (hASH1), encoded by the human ASCL1 gene, belongs to the family of basic helix-loop-helix transcription factors. hASH1 and its mammalian homolog Mash1 are expressed in the central and peripheral nervous system during development, and promote early neuronal differentiation. Furthermore, hASH1 is involved in the specification of neuronal subtype identities. Misexpression of the transcription factor is correlated with a variety of tumors, including lung cancer and neuroendocrine tumors. To gain insights into the molecular mechanisms of hASH1 regulation, we screened for conditions causing changes in hASH1 gene expression rate. We found that treatment of human neuroblastoma-derived Kelly cells with phorbol 12-myristate 13-acetate (PMA) resulted in a fast, strong and long-lasting suppression of hASH1 synthesis. Reporter gene assays with constructs, in which the luciferase activity was controlled either by the ASCL1 promoter or by the hASH1 mRNA untranslated regions (UTRs), revealed a mainly UTR-dependent mechanism. The hASH1 promoter activity was decreased only after 48 h of PMA administration. Our data indicate that different mechanisms acting consecutively at the transcriptional and post-transcriptional level are responsible for hASH1 suppression after PMA treatment. We provide evidence that short term inhibition of hASH1 synthesis is attributed to hASH1 mRNA destabilization, which seems to depend mainly on protein kinase C activity. Under prolonged conditions (48 h), hASH1 suppression is mediated by decreased promoter activity and inhibition of mRNA translation

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

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

Early Exanthema Upon Vemurafenib Plus Cobimetinib Is Associated With a Favorable Treatment Outcome in Metastatic Melanoma: A Retrospective Multicenter DeCOG Study

Background: The combination of BRAF and MEK inhibitors has become standard of care in the treatment of metastatic BRAF V600-mutated melanoma. Clinical factors for an early prediction of tumor response are rare. The present study investigated the association between the development of an early exanthema induced by vemurafenib or vemurafenib plus cobimetinib and therapy outcome. Methods: This multicenter retrospective study included patients with BRAF V600-mutated irresectable AJCC-v8 stage IIIC/D to IV metastatic melanoma who received treatment with vemurafenib (VEM) or vemurafenib plus cobimetinib (COBIVEM). The development of an early exanthema within six weeks after therapy start and its grading according to CTCAEv4.0 criteria was correlated to therapy outcome in terms of best overall response, progression-free (PFS), and overall survival (OS). Results: A total of 422 patients from 16 centers were included (VEM, n=299; COBIVEM, n=123). 20.4% of VEM and 43.1% of COBIVEM patients developed an early exanthema. In the VEM cohort, objective responders (CR/PR) more frequently presented with an early exanthema than non-responders (SD/PD); 59.0% versus 38.7%; p=0.0027. However, median PFS and OS did not differ between VEM patients with or without an early exanthema (PFS, 6.9 versus 6.0 months, p=0.65; OS, 11.0 versus 12.4 months, p=0.69). In the COBIVEM cohort, 66.0% of objective responders had an early exanthema compared to 54.3% of non-responders (p=0.031). Median survival times were significantly longer for patients who developed an early exanthema compared to patients who did not (PFS, 9.7 versus 5.6 months, p=0.013; OS, not reached versus 11.6 months, p=0.0061). COBIVEM patients with a mild early exanthema (CTCAEv4.0 grade 1-2) had a superior survival outcome as compared to COBIVEM patients with a severe (CTCAEv4.0 grade 3-4) or non early exanthema, respectively (p=0.047). This might be caused by the fact that 23.6% of patients with severe exanthema underwent a dose reduction or discontinuation of COBIVEM compared to only 8.9% of patients with mild exanthema. Conclusions: The development of an early exanthema within 6 weeks after treatment start indicates a favorable therapy outcome upon vemurafenib plus cobimetinib. Patients presenting with an early exanthema should therefore be treated with adequate supportive measures to provide that patients can stay on treatment

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

Real-World Therapy with Pembrolizumab: Outcomes and Surrogate Endpoints for Predicting Survival in Advanced Melanoma Patients in Germany

Knowledge on the real-world characteristics and outcomes of pembrolizumab-treated advanced melanoma patients in Germany and on the value of different real-world endpoints as surrogates for overall survival (OS) is limited. A sample of 664 pembrolizumab-treated patients with advanced melanoma from the German registry ADOReg was used. We examined OS, real-world progression-free survival (rwPFS), real-world time to next treatment (rwTtNT), and real-world time on treatment (rwToT). Spearman’s rank and iterative multiple imputation (IMI)-based correlation coefficients were computed between the OS and the rwPFS, rwTtNT, and rwToT and reported for the first line of therapy and the overall sample. The median OS was 30.5 (95%CI 25.0–35.4) months, the rwPFS was 3.9 months (95%CI 3.5–4.9), the rwTtNT was 10.7 months (95%CI 9.0–12.9), and the rwToT was 6.2 months (95%CI 5.1–6.8). The rwTtNT showed the highest correlation with the OS based on the IMI (rIMI = 0.83), Spearman rank correlations (rs = 0.74), followed by the rwToT (rIMI = 0.74 and rs = 0.65) and rwPFS (rIMI = 0.69 and rs = 0.56). The estimates for the outcomes and correlations were similar for the overall sample and those in first-line therapy. The median OS was higher compared to recent real-world studies, supporting the effectiveness of pembrolizumab in regular clinical practice. The rwTtNT may be a valuable OS surrogate, considering the highest correlation was observed with the OS among the investigated real-world endpoints