Molecular Visualization of Neuronal TDP43 Pathology In Situ.

Nuclear exclusion and cytoplasmic accumulation of the RNA-binding protein TDP43 are characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite this, the origin and ultrastructure of cytosolic TDP43 deposits remain unknown. Accumulating evidence suggests that abnormal RNA homeostasis can drive pathological TDP43 mislocalization, enhancing RNA misprocessing due to loss of nuclear TDP43 and engendering a cycle that ends in cell death. Here, we show that adding small monovalent oligonucleotides successfully recapitulates pathological TDP43 mislocalization and aggregation in iPSC-derived neurons (iNeurons). By employing a multimodal in situ cryo-correlative light and electron microscopy pipeline, we examine how RNA influences the localization and aggregation of TDP43 in near-native conditions. We find that mislocalized TDP43 forms ordered fibrils within lysosomes and autophagosomes in iNeurons as well as in patient tissue, and provide the first high-resolution snapshots of TDP43 aggregates in situ. In so doing, we provide a cellular model for studying initial pathogenic events underlying ALS, FTLD, and related TDP43-proteinopathies.

Facile production of tagless membrane scaffold protein for nanodiscs.

The initial step in the preparation of nanodiscs is to express and purify the membrane scaffold protein (MSP) to homogeneity. Current methods used for the isolation and purification of MSP utilize nickel affinity chromatography. However, the presence of a polyhistidine tag on the MSP often interferes with downstream steps where nanodiscs reconstituted with protein need to be isolated from empty ones. Therefore, one must engage in the finicky process of removing the polyhistidine tag from the MSP using a protease before the formation of nanodiscs. Herein, we describe a robust streamlined approach to produce tagless MSP by expression as inclusion bodies followed by cleavage with cyanogen bromide, and purification by gel filtration chromatography. In addition, the MSP prepared is devoid of tryptophan residues which facilitates tryptophan-based spectroscopic studies of reconstituted proteins. Dynamic light scattering and transmission electron microscopy showed that the tagless MSP produced was competent to produce nanodiscs.

Rapid preparation of nanodiscs for biophysical studies.

Nanodiscs, which are disc-shaped entities that contain a central lipid bilayer encased by an annulus of amphipathic helices, have emerged as a leading native-like membrane mimic. The current approach for the formation of nanodiscs involves the creation of a mixed-micellar solution containing membrane scaffold protein, lipid, and detergent followed by a time consuming process (3-12 h) of dialysis and/or incubation with sorptive beads to remove the detergent molecules from the sample. In contrast, the methodology described herein provides a facile and rapid procedure for the preparation of nanodiscs in a matter of minutes (<15 min) using Sephadex® G-25 resin to remove the detergent from the sample. A panoply of biophysical techniques including analytical ultracentrifugation, dynamic light scattering, gel filtration chromatography, circular dichroism spectroscopy, and cryogenic electron microscopy were employed to unequivocally confirm that aggregates formed by this method are indeed nanodiscs. We believe that this method will be attractive for time-sensitive and high-throughput experiments.

Consumo de sustancias psicoactivas en pacientes con trastorno de pánico / Psychoactive drugs use in patients with panic disorder

Introducción. Los objetivos de este estudio fueron evaluar la prevalencia del consumo de drogas en pacientes ambulatorios con trastorno de pánico, su repercusión sobre la evolución y la respuesta al tratamiento de dicho trastorno. Material y métodos. La muestra está constituida por 79 casos con diagnóstico de trastorno de pánico o agorafobia con trastorno de pánico según la CIE-10 y 83 controles con otros trastornos psiquiátricos en tratamiento en el mismo centro. Se realizó un seguimiento durante 6 meses. Resultados. Se obtuvo una prevalencia para el consumo habitual de drogas a lo largo de la vida del 13 % para el alcohol, el 52 % para el tabaco y el 47 % para el café; no hubo consumos de otras sustancias. El consumo actual de cafeína era inferior en los casos que en los controles, no existiendo diferencias respecto a los otros consumos, y el consumo de café se relacionó con requerimientos de mayores dosis de antidepresivos. Conclusiones. Por tanto, la prevalencia de consumo habitual de sustancias a lo largo de la vida en pacientes con trastorno de pánico en tratamiento ambulatorio se sitúa alrededor de un 13 % para el alcohol, un 47 % para el café y un 52 % para el tabaco; hay un menor consumo de cafeína, aunque no existen diferencias en otros consumos respecto a un grupo de controles psiquiátricos. La presencia de agorafobia no repercute en el consumo. El consumo tiene escasas repercusiones sobre la clínica y la respuesta al tratamiento, aunque puede modificar las dosis requeridas de antidepresivos

Introduction. The objectives of this study were to evaluate the prevalence of drug use in out-patients with panic disorder and their influence in evolution and therapeutic response of panic disorder. Material and methods. The sample was made up of 79 out-patients diagnosed of panic disorder or agoraphobia with panic disorder according to the ICD-10 criteria and 83 controls from the same center with other psychiatric disorders. Subjects were followed-up for six months. Results. Prevalence of regular lifetime drug use was: 13 % for alcohol, 52 % for nicotine and 47 % for caffeine. No other drug use was observed. Patients with panic disorder used less caffeine than controls, there being no differences in other drug use. Caffeine use was associated with higher antidepressant dosages. Conclusions. Thus, prevalence of regular drug use in panic disorder during the lifetime of out-patients with panic disorder was: 13 % for alcohol, 47 % for caffeine use and 52 % for nicotine use. Those with panic disorder use less caffeine than other psychiatric patients, but there were no differences in other drug use. Presence of agoraphobia has no repercussion on consumption. There were no differences in clinical manifestations and treatment responses between users and non-users, but drug use may modify antidepressant dosages

[Psychoactive drugs use in patients with panic disorder]. / Consumo de sustancias psicoactivas en pacientes con trastorno de pánico.

INTRODUCTION: The objectives of this study were to evaluate the prevalence of drug use in out-patients with panic disorder and their influence in evolution and therapeutic response of panic disorder. MATERIAL AND METHODS: The sample was made up of 79 out-patients diagnosed of panic disorder or agoraphobia with panic disorder according to the ICD-10 criteria and 83 controls from the same center with other psychiatric disorders. Subjects were followed-up for six months. RESULTS: Prevalence of regular lifetime drug use was: 13 % for alcohol, 52 % for nicotine and 47 % for caffeine. No other drug use was observed. Patients with panic disorder used less caffeine than controls, there being no differences in other drug use. Caffeine use was associated with higher antidepressant dosages. CONCLUSIONS: Thus, prevalence of regular drug use in panic disorder during the lifetime of out-patients with panic disorder was: 13 % for alcohol, 47 % for caffeine use and 52 % for nicotine use. Those with panic disorder use less caffeine than other psychiatric patients, but there were no differences in other drug use. Presence of agoraphobia has no repercussion on consumption. There were no differences in clinical manifestations and treatment responses between users and non-users, but drug use may modify antidepressant dosages.

Efectividad del uso del citalopram en pacientes con trastornos de pánico / Effectiveness of citalopram in out-patients with panic disorder

Nuestro objetivo fue estudiar la efectividad y tolerancia del citalopram en el trastorno de pánico. Cincuenta y cinco pacientes con trastorno de pánico o agorafobia con trastorno de pánico según criterios de la CIE-10 que iniciaron tratamiento ambulatorio, fueron seguidos durante seis meses. La dosis de citalopram se ajustó según criterios clínicos y se pautaron benzodiacepinas si se consideraba que estaban indicadas. Se valoró la efectividad del tratamiento usando como variables dependientes el cambio en la frecuencia de las crisis y en la impresión clínica global del médico (ICG), como variables de efectividad secundarias se usaron los cambios en diversas escalas de depresión y ansiedad. Once pacientes abandonaron el seguimiento. El 83,4% de los pacientes presentó una mejoría marcada o notable según la ICG de eficacia. Los efectos secundarios más frecuentes, fueron las disfunciones sexuales y las molestias abdominales. Por lo tanto, el citalopram puede resultar efectivo en disminuir el número de crisis de angustia y mejora sustancialmente otras manifestaciones clínicas en más del 80% de los pacientes, con un favorable perfil de tolerancia, en las condiciones clínicas habituales

The objective of this study was to study the effectiveness and tolerability of citalopram in panic disorder. Fifty five out-patients with panic disorder or agoraphobia with panic disorder according to the ICD-10 criteria who initiated treatment, were followed up for six months. Dosages of citalopram were ajusted according clinical criteria, and use of benzodiacepines was permitted. Effectiveness variables were the changes in number of panic attacks, and the clinical global impression (CGI) of efficacy, and secondary variables, were the changes of scores in a variety of depressión and anxiety scales. Eleven patients were drop-outs. A 83.4% of patients had a notably or excellent improvement according to the CGI. More prominent adverse effects were sexual dysfunction and gastrointestinal discomfort. Citalopram appears effective to improve the number of panic attacks and another clinical manifestations of panic disorder in 80% of the patients, with a favourable tolerability profile