Elevated mRNA-levels of distinct mitochondrial and plasma membrane Ca(2+) transporters in individual hypoglossal motor neurons of endstage SOD1 transgenic mice.

Tobias Mã¼Hling,Birgit Liss,Albert C Ludolph,Jochen H Weishaupt,Johanna Duda

doi:10.3389/fncel.2014.00353

Tobias Mã¼Hling, Birgit Liss + Show 3 more

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https://doi.org/10.3389/fncel.2014.00353

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Abstract

Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS), a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs) from a common ALS mouse model, the endstage superoxide dismutase SOD1G93A transgenic mouse, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT) positive hMNs from wildtype (WT) and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1, and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na+/Ca2+ exchanger NCX1 were also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS.

Highlights

Amyotrophic Lateral Sclerosis (ALS) is the most common motor neuron (MN) degenerative disease, with an adult onset and an annual incidence of 1–3 cases per 100,000 people worldwide (Ludolph et al, 2012; Valori et al, 2014)
Quantifying NADH dehydrogenase subunit 1 (ND1) DNA copies in parallel with mitochondrial Ca2+ transporter mRNAs in individual hMNs allows the normalization of mitochondrial Ca2+ transporter expression levels to the number of mitochondria/mitochondrial genomes in the respective analyzed hMN pools
By cell-specific normalization of hMN Quantitative PCR (qPCR) data for all analyzed plasma membrane Ca2+ transporters to relative ChAT levels, we detected about 1.7-fold higher mRNA-levels, selectively of NCX1 in hMNs from SOD1G93A mice compared to WT (Figure 4B right and Table 2B)

Summary

Introduction

Amyotrophic Lateral Sclerosis (ALS) is the most common motor neuron (MN) degenerative disease, with an adult onset and an annual incidence of 1–3 cases per 100,000 people worldwide (Ludolph et al, 2012; Valori et al, 2014). There is a family history in around 10% of ALS patients, that can be attributed to several gene defects (Renton et al, 2013). Most common is an abnormal hexanucleotide expansion of the chromosome 9 open reading frame 72 gene (C9ORF72), present in many familial as well as some sporadic ALS cases (DeJesus-Hernandez et al, 2011; Renton et al, 2011; Haeusler et al, 2014). ALS patients and its transgenic animal models, there is evidence of ubiquitinated protein inclusions in MNs as well as Frontiers in Cellular Neuroscience www.frontiersin.org

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