Elsevier

Neurobiology of Disease

Volume 103, July 2017, Pages 1-10
Neurobiology of Disease

Evidence that increased Kcnj6 gene dose is necessary for deficits in behavior and dentate gyrus synaptic plasticity in the Ts65Dn mouse model of Down syndrome

https://doi.org/10.1016/j.nbd.2017.03.009Get rights and content

Highlights

  • A role for Kcnj6 gene dose in Down syndrome was examined using Ts65Dn mice.

  • Reduction in Kcnj6 gene dose to normal restored hippocampal level of Kir3.2.

  • Reduction in Kcnj6 gene dose restored memory in the novel object recognition test.

  • Reduction in Kcnj6 gene dose restored STP and LTP in dentate gyrus.

  • Suppression of Kir3.2 channels with fluoxetine restored STP and LTP in DG.

Abstract

Down syndrome (DS), trisomy 21, is caused by increased dose of genes present on human chromosome 21 (HSA21). The gene-dose hypothesis argues that a change in the dose of individual genes or regulatory sequences on HSA21 is necessary for creating DS-related phenotypes, including cognitive impairment. We focused on a possible role for Kcnj6, the gene encoding Kir3.2 (Girk2) subunits of a G-protein-coupled inwardly-rectifying potassium channel. This gene resides on a segment of mouse Chromosome 16 that is present in one extra copy in the genome of the Ts65Dn mouse, a well-studied genetic model of DS. Kir3.2 subunit-containing potassium channels serve as effectors for a number of postsynaptic metabotropic receptors including GABAB receptors. Several studies raise the possibility that increased Kcnj6 dose contributes to synaptic and cognitive abnormalities in DS. To assess directly a role for Kcnj6 gene dose in cognitive deficits in DS, we produced Ts65Dn mice that harbor only 2 copies of Kcnj6 (Ts65Dn:Kcnj6 ++− mice). The reduction in Kcnj6 gene dose restored to normal the hippocampal level of Kir3.2. Long-term memory, examined in the novel object recognition test with the retention period of 24 h, was improved to the level observed in the normosomic littermate control mice (2N:Kcnj6 ++). Significantly, both short-term and long-term potentiation (STP and LTP) was improved to control levels in the dentate gyrus (DG) of the Ts65Dn:Kcnj6 ++− mouse. In view of the ability of fluoxetine to suppress Kir3.2 channels, we asked if fluoxetine-treated DG slices of Ts65Dn:Kcnj6 +++ mice would rescue synaptic plasticity. Fluoxetine increased STP and LTP to control levels. These results are evidence that increased Kcnj6 gene dose is necessary for synaptic and cognitive dysfunction in the Ts65Dn mouse model of DS. Strategies aimed at pharmacologically reducing channel function should be explored for enhancing cognition in DS.

Introduction

Down syndrome (DS), due to triplication of human chromosome 21 (HSA21) (Lejeune et al., 1959), results in a number of significant neurobiological and somatic phenotypes. The most salient are cognitive and behavioral impairments in children and the emergence of Alzheimer's disease in the elderly (Belichenko et al., 2016, Dierssen, 2012, Dykens, 2007, Kleschevnikov et al., 2012c, Lott, 2012, Nadel, 2003, Roizen and Patterson, 2003, Roper and Reeves, 2006, Sabbagh and Edgin, 2016). The gene-dosage hypothesis proposes that all DS-related phenotypes are due to the presence in excess of one or more genes or regulatory sequences on HSA21 (Epstein et al., 1981). Tests of this hypothesis has in recent years resulted in insights into a number of the genes and mechanisms responsible for DS phenotypes (Belichenko et al., 2009, Dierssen et al., 2011, Fotaki et al., 2002, Kleschevnikov et al., 2004, Olson et al., 2004b, Salehi et al., 2006, Vesa et al., 2005).

Mouse genetic models of DS provide an opportunity to investigate the neurobiology of DS (Belichenko et al., 2015, Das and Reeves, 2011, Edgin et al., 2012, Gardiner et al., 2003, Mojabi et al., 2016, Reeves et al., 1995, Rueda et al., 2012, Yu et al., 2010) and to explore the role of individual genes in DS phenotypes (Altafaj et al., 2013, Salehi et al., 2006). Similar to DS, mouse genetic models of DS exhibit deficient hippocampus-dependent long-term memory, working memory, and other physiological and behavioral changes consistent with deficits in cognition (Fernandez et al., 2007). Mouse models that contain different as well as overlapping sets of triplicated genes have been genetically engineered and examined to explore phenotype-genotype relationship (Davisson et al., 1990, Jiang et al., 2015, O'Doherty et al., 2005, Olson et al., 2004a, Pereira et al., 2009, Sago et al., 1998, Yu et al., 2010). The most intensively investigated mouse is the Ts65Dn model, whose creation 26 years ago was instrumental in advancing this field of study (Davisson et al., 1990). Ts65Dn mice exhibit many phenotypes characteristic of DS including deficient cognition (Braudeau et al., 2011, Busciglio et al., 2013, Costa, 2011, Demas et al., 1998, Faizi et al., 2011, Fernandez et al., 2007, Gutierrez-Castellanos et al., 2013, Martinez-Cue et al., 2014, Smith et al., 2014).

Studies in humans partially trisomic for HSA21 identified a region whose presence in three copies was sufficient to create many DS phenotypes, including intellectual disability; it was thereby labeled the Down Syndrome Critical Region (DSCR) (Korenberg, 1990, Peterson et al., 1994, Rahmani et al., 1989, Yamamoto et al., 2011). Recent studies in mice carrying all or part of the homologous region of mouse Chromosome 16 have shown that many DS-like phenotypes are linked to genes in this region (Belichenko et al., 2009, Dierssen and de Lagran, 2006, Jiang et al., 2015, Reeves et al., 1995). Within the DSCR, Kcnj6 is a candidate for contributing through increased dose to cognitive deficits. Kcnj6 is present in 3 copies in both people with DS and Ts65Dn mice. This gene encodes the Kir3.2 (Girk2) subunit of inwardly rectifying potassium channels which serve as effectors for a number of postsynaptic metabotropic receptors (Luscher et al., 1997, Mark and Herlitze, 2000, Yamada et al., 1998). As predicted by increased dose, the Kir3.2 product of Kcnj6 is increased in Ts65Dn mice (Harashima et al., 2006, Kleschevnikov et al., 2012b, Kleschevnikov et al., 2005). Suggesting a physiologically meaningful contribution for increased Kir3.2 in these mice, there was increased signaling through postsynaptic GABAB receptors in both primary cultures of hippocampal neurons (Best et al., 2007) and acute hippocampal slices (Best et al., 2012, Kleschevnikov et al., 2012b). In addition, suppressing enhanced GABAB/Kir3.2 signaling by treating with selective GABAB receptor antagonists restored synaptic plasticity and long-term memory in Ts65Dn mice (Kleschevnikov et al., 2012a). Recently, cognitive assessment in a series of mouse genetic models bearing distinct sets of genes within DSCR pointed to a contribution of Kcnj6 (Jiang et al., 2015).

A direct test of the impact of increased dose of specific genes is essential for defining contribution(s) to phenotypes. To address the impact of Kcnj6 triplication on cognitive phenotypes in DS, we genetically deleted the third copy by producing Ts65Dn mice with 2 copies of Kcnj6 (i.e., Ts65Dn:Kcnj6 ++−). Littermate Ts65Dn mice with three copies of Kcnj6 (Ts65Dn:Kcnj6 +++) and normosomic mice (2N:Kcnj6 ++) served as controls. Reduction of the Kcnj6 gene dose restored to normal the level of Kir3.2, long term memory, and short- and long- term potentiation in the DG. Remarkably, pharmacologically inhibiting Kir3.2-containing channels also restored synaptic plasticity. The findings are evidence that increased expression of Kcnj6 is necessary for the significant cognitive impairment in this model of DS and suggests that strategies aimed at pharmacologically reducing channel function should be explored for enhancing cognition in DS.

Section snippets

Animals

Segmental trisomy 16 (Ts65Dn) mice were purchased from the Jackson Laboratory, Bar Harbor, ME, stock #001924. Heterozygous Kcnj6 +/− mice (129/sv–C57BL/6 hybrid crosses) were a gift from Dr. L. Jan (UCSF). These mice were characterized in a number of previous studies (Blednov et al., 2001, Mitrovic et al., 2003, Signorini et al., 1997). Ts65Dn:Kcnj6 ++− mice were the result of crossing female Ts65Dn with male Kcnj6 +/− mice. Diploid (2N:Kcnj6 ++) and trisomic (Ts65Dn:Kcnj6 +++) mice served as

Kcnj6 dose influences body weight during aging

Body weight is a measure of systemic health. Children with DS show reduced body weight (Cronk, 1978), while adult individuals with DS often have increased body weight due to obesity (de Asua et al., 2014). To assess for an effect of Kcnj6 gene dose, the body weights of mice of each genotype were examined from 1 to 12 months of age (Fig. 1). Body weight was significantly reduced in Ts65Dn:Kcnj6 +++ vs. 2N:Kcnj6 ++ mice at all ages (Fig. 1, red vs. blue bar respectively; Table 1). Interestingly,

Discussion

Herein we examined the effects of the Kcnj6 gene dose on behavioral and cognitive phenotypes in the Ts65Dn mouse model of DS. Genetically normalizing Kcnj6 gene dose reduced to 2N levels the Kir3.2 protein subunit of inwardly-rectifying potassium channels, restored synaptic plasticity in the DG, and improved long-term memory. Our findings are evidence that increased gene dose for Kcnj6 is necessary for deficits in hippocampal synaptic plasticity and hippocampally-mediated tests of long-term

Acknowledgements

The study supported by The Jerome Lejeune Foundation (Grant # 1483), Cure Alzheimer's Foundation, LuMind /RDS Foundation, and NIH (R01NS55371 and R01NS66072).

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