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

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

Charakterisierung der splicevarianten-spezifischen Effekte des RNA-editierten Glycinrezeptors alpha 3 im Hippocampus

Previous studies showed that RNA processing (editing and splicing) of GlyRs is changed in hippocampus of patients with temporal lobe epilepsy (TLE). RNA edited GlyR α3K was shown to be involved in tonic inhibition and neurodegeneration, while the RNA edited long splice variant GlyR α3L was postulated to exert a functional role at glutamatergic synapses (Eichler et al., 2008; Eichler et al., 2009). Furthermore, it was shown the potassium- chloride cotransporter 2 (KCC2) protects neurons against neurodegeneration mediated by the RNA edited short GlyR α3K185L variant. These results indicated that RNA processing of GlyR α3 and regulation of intracellular chloride may be important factors of TLE. Aim of this study was to characterize splice variant-specific effects of RNA-edited GlyR α3185L by identifying mechanisms responsible for GlyR α3K185L-dependent neurodegeneration and KCC2-dependent neuroprotection and investigating the functional impact of GlyR α3L185L on glutamatergic synaptic transmission in vivo. My study revealed that GlyR α3K185L activation changes neuronal membrane properties (membrane resistance) and thereby induces neurodegeneration. Furthermore, KCC2-mediated neuroprotection was found to depend on a structural role of KCC2 rather than involving regulation of intracellular chloride. My study also revealed that the RNA splice insert of the long GlyR α3L subunit interacts with Sec8, a component of the exocyst complex of vesicular trafficking factors, and leads to axonal expression and presynaptic localization of GlyR α3L185L. The presynaptic mode of action of this RNA-edited GlyR α3L variant facilitates synaptic transmission in vivo, and depending on the neuron type in which it was expressed, quite different phenotypes of the corresponding knockin animals were observed. Enhanced glutamatergic synaptic transmission resulted in impaired working and reference memories and cognitive dysfunction, whereas anxiety-related behavior resulted from GlyR α3L185L expression in presynaptic terminals of parvalbumin-positive interneurons. Thus, my study identifies a new structural role for KCC2 in neuroprotection, and it shows that splice variant-specific effects of RNA-edited GlyR α3185L can contribute to the psychopathology of TLE by triggering neurodegeneration (α3K185L) and cognitive dysfunction as well as anxiety (α3L185L), which are well established neuropsychiatric symptoms of TLE.Frühere Studien zeigten, dass die GlyR RNA-Prozessierung (Editierung und Spleißen) in den Hippocampi von Temporallappenepilesie (TLE) Patienten verändert ist. Es ist bekannt, dass die RNA-editierte kurze α3K-GlyR Spleißvariante tonische Inhibition und Neurodegeneration vermittelt, während die RNA-editierte lange α3L-GlyR Variante eine Rolle bei der glutamatergen synaptischen Transmission zu spielen scheint (Eichler et al., 2008; Eichler et al., 2009). Desweiteren wurde gezeigt, dass der Kalium-Chlorid Cotransporter 2 (KCC2) Neurone vor GlyR α3K185L-vermittelter Neurodegeneration schützt. Diese Ergebnisse deuten an, dass sowohl RNA Prozessierung der GlyR α3-Untereinheit als auch die Regulation des intrazellulären Chloridspiegels krankheitsbestimmende Faktoren der TLE sind. Deshalb war das Ziel meiner Arbeit, Spleißvarianten-spezifische Effekte der RNA-editierten GlyR α3-Untereinheit zu charakterisieren. Hierfür sollten frühere Studien zur neurodegenerativen Rolle des α3K185L-GlyR vertieft werden und ein besonderes Augenmerk auf die Mechanismen der KCC2-vermittelten Neuroprotektion gerichtet werden. Andererseits sollte die Untersuchung der funktionellen Auswirkungen von α3L185L-GlyR auf synaptische Transmission und Verhalten das Verständnis von pathogenen GlyR-spezifischen RNA-Mechanismen vertiefen. Meine Arbeit zeigt, dass die Rezeptoraktivierung Änderungen in den Membraneigenschaften (Membranwiderstand) hervorruft und dadurch Neurodegeneration triggert. Im Gegensatz zu vorherigen Annahmen ergaben meine Untersuchungen jedoch, dass die KCC2 vermittelte Neuroprotektion nicht vom Chloridtransport abhängt, sondern vielmehr eine strukturelle Funktion des KCC2-Proteins involviert. Meine Studien zeigten ferner, dass das RNA-Spleißinsert der GlyR α3L-Variante mit dem Sec8-Protein der Exozyst-Komplex-Proteinfamilie von vesikulären Zielsteuerungsfaktoren interagiert, wodurch es zur axonalen und präsynaptischen Rezeptorexpression kommt. Dort übt die RNA-editierte α3L185L- Rezeptorvariante fazilitierende Effekte auf die Neurotransmitterfreisetzung aus. In vivo äußert sich dies in unterschiedlichen Phänotypen der korrespondierenden genetisch veränderten Mäuse, die vom betroffenen Neuronentyp abhängen. Die verstärkte glutamaterge Transmission ruft Beeinträchtigungen des Kurz- und Langzeitgedächtnisses sowie kognitive Dysfunktion hervor, wohingegen die gesteigerte GABAerge synaptische Transmission an parvalbumin-enthaltenden synaptischen Endknöpfchen Angstverhalten fördert

PREVALÊNCIA DE TRANSTORNOS RESPIRATÓRIOS DO SONO EM PORTADORES DE DIABETES MELLITUS TIPO 2

Estimar a prevalência de transtornos respiratórios do sono (TRS) em diabéticos mellitus tipo 2 pertencentes ao programa de saúde da família de Ijuí/ RS, assim como verificar a correlação destes com medidas antropométricas, capacidade funcional e qualidade de vida (QV). Trata-se de um estudo transversal que analisou medidas antropométricas, fatores de risco cardiovascular, risco de TRS, sonolência diurna, capacidade funcional e QV. Participaram do estudo 16 indivíduos sendo a maioria mulheres (69%) com média de idade 65 ± 11 anos. A maioria dos indivíduos apresentou alto risco de ter TRS (81%) e 31% apresentam sonolência diurna. Os indivíduos possuem boa capacidade funcional (102% do previsto) e boa QV (94,6 ± 8,4). A sonolência diurna teve correlação negativa com a idade (r=-0,5 e p=0,04). Não foram encontradas outras correlações. Os indivíduos deste estudo têm alto risco de ter TRS sendo recomendada uma avaliação mais criteriosa através da polissonografia

RELAÇÃO DAS MEDIDAS ANTROPOMÉTRICAS COM A PRÁTICA DE ATIVIDADE FÍSICA NÃO ORIENTADA EM INDIVÍDUOS COM DIABETES TIPO 2

Objetivo: avaliar a influência da prática de atividade física não orientada sobre as medidas antropométricas em indivíduos com diabetes tipo 2. Metodologia: estudo transversal e analítico aprovado pelo CEP UNIJUI nº 91/2010. A amostra foi composta por 178 diabéticos do meio urbano de Ijuí e cadastrados em estratégia de saúde da família (ESF). Os participantes obtiveram o perfil clínico traçado para identificar condições de saúde e medidas antropométricas. Foram questionados a respeito da prática de atividade física não orientada. Para análise dos dados utilizado SPSS 18.0 (Chicago, IL) e a normalidade das variáveis foi avaliada através do teste de Kolmogorov-Smirnov e o teste de correlação de Spearmann foi utilizado para correlação de variáveis não paramétricas. Resultados: a idade média foi de 61,8 anos e a maioria da amostra foi composta por mulheres (63,5%), idosos (60,1%) e sedentários (55,6%). Apresentaram massa corporal 78,9±15,1 kg, estatura 1,6±0,1 cm, IMC 30,6±5,6 kg/m², relação cintura/quadril 1,0±0,08 cm, braço direito 32,8±4,1 cm e braço esquerdo 32,3±4,6 cm. Nenhuma das medidas antropométricas obtiveram relação positiva com a prática de atividade física não orientada (p>0,05). Conclusão: Não foi encontrada nenhuma relação estatisticamente significativa entre as variáveis analisadas, porém ao comparar os grupos, os que praticam atividade física apresentaram massa corporal, circunferência da cintura e quadril discretamente mais baixos

Identification of a new genomic hot spot of evolutionary diversification of protein function.

Establishment of phylogenetic relationships remains a challenging task because it is based on computational analysis of genomic hot spots that display species-specific sequence variations. Here, we identify a species-specific thymine-to-guanine sequence variation in the Glrb gene which gives rise to species-specific splice donor sites in the Glrb genes of mouse and bushbaby. The resulting splice insert in the receptor for the inhibitory neurotransmitter glycine (GlyR) conveys synaptic receptor clustering and specific association with a particular synaptic plasticity-related splice variant of the postsynaptic scaffold protein gephyrin. This study identifies a new genomic hot spot which contributes to phylogenetic diversification of protein function and advances our understanding of phylogenetic relationships

Presentation1.pdf

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