RCAN1 Knockdown Reverts Defects in the Number of Calcium-Induced Exocytotic Events in a Cellular Model of Down Syndrome.

Jacqueline Vásquez-Navarrete,Ximena Baéz-Matus,Sebastián Brauchi,Ana M Cárdenas,Stéphane Ory,Agustín D Martínez,Pablo Caviedes,Arlek M González-Jamett

doi:10.3389/fncel.2018.00189

Abstract

In humans, Down Syndrome (DS) is a condition caused by partial or full trisomy of chromosome 21. Genes present in the DS critical region can result in excess gene dosage, which at least partially can account for DS phenotype. Although regulator of calcineurin 1 (RCAN1) belongs to this region and its ectopic overexpression in neurons impairs transmitter release, synaptic plasticity, learning and memory, the relative contribution of RCAN1 in a context of DS has yet to be clarified. In the present work, we utilized an in vitro model of DS, the CTb neuronal cell line derived from the brain cortex of a trisomy 16 (Ts16) fetal mouse, which reportedly exhibits acetylcholine release impairments compared to CNh cells (a neuronal cell line established from a normal littermate). We analyzed single exocytotic events by using total internal reflection fluorescence microscopy (TIRFM) and the vesicular acetylcholine transporter fused to the pH-sensitive green fluorescent protein (VAChT-pHluorin) as a reporter. Our analyses showed that, compared with control CNh cells, the trisomic CTb cells overexpress RCAN1, and they display a reduced number of Ca2+-induced exocytotic events. Remarkably, RCAN1 knockdown increases the extent of exocytosis at levels comparable to those of CNh cells. These results support a critical contribution of RCAN1 to the exocytosis process in the trisomic condition.

Highlights

Down Syndrome (DS), a condition caused by the presence of an extra copy of chromosome 21, is manifested by multiple abnormalities, the most prominent features being neurological and cognitive disabilities
The role of Rcan1 in this dysfunction, we monitored exocytosis in CTb cells expressing the vesicular acetylcholine transporter fused to the pH-sensitive green fluorescent protein (VAChTpHluorin) by total internal reflection fluorescence microscopy (TIRFM)
We found that CTb cells overexpress Rcan1 and exhibit a reduced number of Ca2+-induced exocytosis events, compared to CNh cells, a cell line established from a normal littermate

Summary

Introduction

Down Syndrome (DS), a condition caused by the presence of an extra copy of chromosome 21, is manifested by multiple abnormalities, the most prominent features being neurological and cognitive disabilities. Reduced levels of some neurotransmitters, such as glutamate, GABA, dopamine and serotonin have been found in brains of DS individuals (Godridge et al, 1987; Reynolds and Warner, 1988; Risser et al, 1997). These alterations in neurotransmission could in turn impair synaptic plasticity (Siarey et al, 1997, 1999, 2005; Kleschevnikov et al, 2004; Belichenko et al, 2007), and hinder learning and memory (Morice et al, 2008)

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