Elsevier

Experimental Neurology

Volume 201, Issue 1, September 2006, Pages 182-192
Experimental Neurology

Potassium channel dysfunction and depolarized resting membrane potential in a cell model of SCA3

https://doi.org/10.1016/j.expneurol.2006.03.029Get rights and content

Abstract

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat within the disease protein, ataxin-3. There is growing evidence that neuronal electrophysiological properties are altered in a variety of polyglutamine diseases such as Huntington's disease and SCA1 and that these alterations may contribute to disturbances of neuronal function prior to neurodegeneration. To elucidate possible electrophysiological changes in SCA3, we generated a stable PC12 cell model with inducible expression of normal and mutant human full-length ataxin-3 and analyzed the electrophysiological properties after induction of the recombinant ataxin-3 expression. Neuronally differentiated PC12 cells expressing the expanded form of ataxin-3 showed significantly decreased viabilities and developed ultrastructural changes resembling human SCA3. Prior to neuronal cell death, we found a significant reduction of the resting membrane potential and a hyperpolarizing shift of the activation curve of the delayed rectifier potassium current. These findings indicate that electrophysiological properties are altered in mutant ataxin-3 expressing neuronal cells and may contribute to neuronal dysfunction in SCA3.

Introduction

Spinocerebellar ataxia type 3 (SCA3) or Machado–Joseph disease is the most common autosomal dominantly inherited cerebellar ataxia and is characterized by a progressive cerebellar syndrome accompanied by a variable extent of other neurological symptoms like opthalmoplegia, extrapyramidal symptoms, pyramidal symptoms and polyneuropathy. Neuropathological examination reveals neurodegeneration in specific brain regions as the dentate nucleus of the cerebellum, spinocerebellar tracts and different parts of the brain stem. SCA3 is caused by an unstable CAG repeat expansion in the SCA3 gene leading to an expansion of polyglutamines in the corresponding protein, ataxin-3. SCA3 belongs to the group of polyglutamine (polyQ) diseases which include Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA) and the spinocerebellar ataxias SCA1, SCA2, SCA6, SCA7 and SCA17 (Zoghbi and Orr, 2000). These disorders share important pathological features, such as a progressive phenotype, selective neurotoxicity despite widespread expression of the disease proteins, conformational change of the mutant proteins and accumulation of the mutant protein in ubiquitinated nuclear inclusions (DiFiglia et al., 1997, Paulson et al., 1997, Skinner et al., 1997).

To understand the pathophysiological role of the polyglutamine expansions, different transgenic mouse models displaying neurological and neuropathophysiological abnormalities have been established. Surprisingly, HD transgenic (Mangiarini et al., 1996) and HD knock-in mice (Shelbourne et al., 1999) as well as SCA1 knock-in mice (Lorenzetti et al., 2000) show a neurological phenotype without significant neuronal cell loss. Transgenic SCA1 mice (Clark et al., 1997) become clearly symptomatic before the onset of neurodegeneration. In a conditional HD mouse model, motor functions improved after switching off the transgene expression (Yamamoto et al., 2000). These findings support the hypothesis that in early stages of polyQ diseases neurological and neuropathological symptoms are rather due to neuronal dysfunction than to neuronal cell loss.

Indeed, studies of HD mouse models have shown numerous neurophysiological abnormalities. Among these are (1) NMDA receptor hyperactivity both in the hippocampus and in striatal neurons (Hodgson et al., 1999, Laforet et al., 2001), (2) lack of long-term potentiation in hippocampal synapses (Hodgson et al., 1999, Murphy et al., 2000), (3) decrease in excitatory striatal inputs (Klapstein et al., 2001, Laforet et al., 2001) and (4) changes in passive membrane properties like a depolarized resting membrane potential (RMP) and a higher input resistance, alterations of the action potential and a reduction of both inwardly and outwardly rectifying K+ currents in striatal spiny neurons (Klapstein et al., 2001, Ariano et al., 2005).

Electrophysiological abnormalities have also been described in SCA models. In SCA6, a variety of alterations of the P/Q-calcium channel function have been found (Piedras-Renteria et al., 2001, Restituito et al., 2000, Toru et al., 2000). Since the SCA6 gene encodes the pore-forming subunit (α1A subunit) of the P/Q-calcium channel (Zhuchenko et al., 1997), SCA6 exhibits features of an ion channel disease which may not be representative for other polyglutamine diseases.

In a transgenic SCA1 mouse, a higher input resistance and a higher A-like potassium conductance in Purkinje cells and a secondary calcium increase following climbing fiber stimulation were found (Inoue et al., 2001). In addition, gene expression profiling in three SCA1 transgenic mouse lines linked a specifically altered set of genes involved in glutamate signaling in Purkinje cells to SCA1 pathophysiology (Yue et al., 2001).

Thus, there is growing evidence that neuronal electrophysiological properties are altered in a variety of polyQ diseases and that these alterations may contribute to disturbances of neuronal function possibly leading to the development of neurological symptoms in the early stage of the diseases prior to neuronal cell loss.

In this study, we generated a stable PC12 cell model of SCA3 with inducible expression of normal and mutant human full-length ataxin-3 and analyzed the electrophysiological properties of these cells. In neuronally differentiated PC12 cells expressing mutant ataxin-3, we found a significant reduction of the resting membrane potential and a hyperpolarizing shift of the activation curve of the delayed rectifier K+ channel. These electrophysiological alterations may contribute to neuronal dysfunction in SCA3.

Section snippets

Generation and characterization of inducible PC12 cell lines

For stable transfection, the recently described tetracycline response element (TRE) containing SCA3 cDNA constructs encoding either human full-length ataxin-3 with 23 (pUHD-SCA3-Q23) or 70 (pUHD-SCA3-Q70) CAG repeats (Evert et al., 1999) and the corresponding empty plasmid pUHD10-3 (Gossen and Bujard, 1992) as mock control were used. Ten micrograms of each construct was co-electroporated with 2 μg pTK-Hyg (Clontech, Palo Alto, CA) into PC12 Tet-on cells (Clontech) with a gene pulser (Biorad,

PC12 cell lines expressing human ataxin-3

The time course of ataxin-3 expression was analyzed after 3, 5, 8, 10 and 12 days in the uninduced (− Dox), induced (+ Dox) and induced/differentiated condition (+ Dox/+ NGF) in stable inducible PC12 Tet-on cell lines expressing human full-length ataxin-3 with 23 (SCA3-Q23) or 70 (SCA3-Q70) repeats. In the absence of doxycycline, transgene expression was almost not detectable over the time course while endogenous rat ataxin-3 was consistently detected at 45 kDa in all selected clonal cell lines at

Discussion

In the present study, we generated a stable PC12 cell model inducibly expressing either normal or mutant human full-length ataxin-3. Induced expression of mutant ataxin-3 in neuronally differentiated cells led to a constant decline of metabolically active cells and was associated ultrastructurally by the formation of nuclear inclusions already after 5 days of induced expression. Prolonged expression resulted in significantly decreased viabilities and severe ultrastructural changes of the

Acknowledgments

We thank H. Beck for critically reading the manuscript and helpful discussions. The excellent technical assistance of C. Kindermann and A.M. Vieira-Saecker is gratefully acknowledged. This work was supported by the European Union (EU, 6th framework, EuroSCA TP3). A.S. and M.H were supported by a University of Bonn Center Grant (BONFOR).

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