Elevated alpha-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson´s disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson´s disease progression, particularly in the context of bioenergetic dysfunction.Fil: Oliveira, L. M. A.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Falomir Lockhart, Lisandro Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentina. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Botelho, M. G.. Max-Planck-Institut für biophysikalische Chemie; Alemania. Universidade Federal do Rio de Janeiro; BrasilFil: Lin, K. H.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Wales, P.. Universität Göttingen; AlemaniaFil: Koch, J. C.. Universität Göttingen; AlemaniaFil: Gerhardt, Elizabeth. Universität Göttingen; AlemaniaFil: Taschenberger, H.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Outeiro, T. F.. Universität Göttingen; AlemaniaFil: Lingor, P.. Universität Göttingen; AlemaniaFil: Schüele, B.. The Parkinson’s Institute; Estados UnidosFil: Arndt Jovin, D. J.. Max-Planck-Institut für biophysikalische Chemie; AlemaniaFil: Jovin, T. M.. Max-Planck-Institut für biophysikalische Chemie; Alemani

Dependence of α-synuclein aggregate morphology on solution conditions

α-Synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from α-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type α-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl, were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalene-sulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating α-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions similar to100 mum) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2 M NaCl or 10 mM MgCl, to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of α-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions. (C) 2002 Elsevier Science Ltd. All rights reserved

Imaging the Electrocyte of Torpedo Marmorata by Scanning Force Microscopy

Scanning force microscopy (SFM) and scanning electron microscopy (SEM) were used to examine the tissue structure of the electric organ of Torpedo marmorata in air and in liquid after applying fracturing and cryosectioning techniques and chemical fixation. The electric organ is organized in columns of stacked electrocytes, arranged in a honeycomb pattern. The columns were cut along a plane normal to the cell stack and thin sections were transferred to polylysine coated glass coverslips. The polarity of the electrocytes was made apparent by immunofluorescence microscopy directed to different domains of the acetylcholine receptor (AChR), thus revealing the innervated face of the cell. SFM and SEM both showed cell surfaces to be overlaid by a network of collagen fibers by their characteristic banding pattern with about 64 nm periodicity and about 2.5 nm corrugation amplitude. In liquid, significantly lower structural resolution was achieved by SFM, probably due to sample elasticity