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

Mühling T, Duda J, Weishaupt JH, Ludolph AC, Liss B - Front Cell Neurosci (2014)

Bottom Line: This functional deficit was defined by a reduced hMN mitochondrial Ca(2+) uptake capacity and elevated Ca(2+) extrusion across the plasma membrane.These higher expression-levels of mitochondrial Ca(2+) transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification.Thus, pharmacological stimulation of Ca(2+) transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physiology, Institute of Applied Physiology, Ulm University Ulm, Germany.

ABSTRACT
Disturbances in Ca(2+) 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 Ca(2+) homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs) from a common ALS mouse model, the endstage superoxide dismutase SOD1(G93A) transgenic mouse, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca(2+) uptake capacity and elevated Ca(2+) extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca(2+) transporters in individual, choline-acetyltransferase (ChAT) positive hMNs from wildtype (WT) and endstage SOD1(G93A) 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 SOD1(G93A) mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca(2+) transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca(2+) transporters MCU/MICU1, Letm1, and UCP2 in remaining hMNs from endstage SOD1(G93A) mice. These higher expression-levels of mitochondrial Ca(2+) 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(+)/Ca(2+) exchanger NCX1 were also about 2-fold higher in hMNs from SOD1(G93A) mice. Thus, pharmacological stimulation of Ca(2+) transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS.

No MeSH data available.


Related in: MedlinePlus

Elevated mRNA-levels of distinct Ca2+ transporters in individual hypoglossal motor neurons from SOD1G93A mice compared to WT. (A) Cell-specific RT-qPCR data for mitochondrial and plasma membrane Ca2+ transporters, derived from pools of 15 hMNs each from SOD1G93A mice and WT. Data are given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse brainstem tissue. (B) Data from (A), normalized to mitochondrially coded ND1 DNA-levels for mitochondrial Ca2+ transporters, and to ChAT cDNA levels for plasma membrane Ca2+ transporters. Significant differences according to Mann-Whitney-U-Tests are marked with (*), as defined in methods section. Note that data in (A,B) for the respective genes refer to two different axes, as indicated by the dashed line. (C) Right: Overview of concerted elevated Ca2+ transporter expression in individual hMN from SOD1G93A mice compared to WT (marked in red: MCU/MICU1, Letm1, UCP2, and NCX1). Left: These findings can provide a molecular basis for the recently described, selective functional, activity-dependent Ca2+ homeostasis deficit in hMN from endstage SOD1G93A mice. Insert shows stimulation protocol and resulting electrophysiological recordings (scale bar: 1 s), combined with calcium imaging (for details please see text and Fuchs et al., 2013).
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Figure 4: Elevated mRNA-levels of distinct Ca2+ transporters in individual hypoglossal motor neurons from SOD1G93A mice compared to WT. (A) Cell-specific RT-qPCR data for mitochondrial and plasma membrane Ca2+ transporters, derived from pools of 15 hMNs each from SOD1G93A mice and WT. Data are given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse brainstem tissue. (B) Data from (A), normalized to mitochondrially coded ND1 DNA-levels for mitochondrial Ca2+ transporters, and to ChAT cDNA levels for plasma membrane Ca2+ transporters. Significant differences according to Mann-Whitney-U-Tests are marked with (*), as defined in methods section. Note that data in (A,B) for the respective genes refer to two different axes, as indicated by the dashed line. (C) Right: Overview of concerted elevated Ca2+ transporter expression in individual hMN from SOD1G93A mice compared to WT (marked in red: MCU/MICU1, Letm1, UCP2, and NCX1). Left: These findings can provide a molecular basis for the recently described, selective functional, activity-dependent Ca2+ homeostasis deficit in hMN from endstage SOD1G93A mice. Insert shows stimulation protocol and resulting electrophysiological recordings (scale bar: 1 s), combined with calcium imaging (for details please see text and Fuchs et al., 2013).

Mentions: Next, via RT-qPCR, we analyzed qualitative mRNA expression of the main described potential mitochondrial and plasma membrane Ca2+ transporters (Figure 3A) in pools of 15 hMNs from WT and SOD1G93A mice. As illustrated in Figure 3B, mRNAs for all tested mitochondrial Ca2+ transporters (MCU/MICU1/MCUR1, Letm1, UCP2, MNCX) but UCP3, and for all plasma membrane Ca2+ transporters (PMCA1-4, NCX1, NCX3) but NCX2, were detected in ChAT and ND1 positive hMNs (n = 3 pools of 15 neurons each) from WT and SOD1G93A mice. Accordingly, we quantified mRNA-levels for all these mitochondrial and plasma membrane Ca2+ transporters via qPCR in hMNs from WT and endstage SOD1G93A mice. Results are given in Figure 4 and Table 2B. Without cell-specific normalization, we detected significantly higher mRNA levels of only MICU1 and UCP2 in hMNs from SOD1G93A mice (each about 1.7-fold higher compared to WT; Figure 4A left and Table 2B). All other Ca2+ transporter mRNA-levels were not significantly altered (although trends were observed in particular for MCU and Letm1, compare Figure 4A and Table 2B). Accordingly, cell-specific normalization of hMN qPCR data for mitochondrial Ca2+ transporters to mitochondrially coded ND1 DNA-levels (and thus to respective hMN mitochondrial genome numbers) revealed significantly higher mRNA levels of not only MICU1 and UCP2, but also of MCU and Letm1 in hMNs from SOD1G93A mice (each about 1.8-fold higher compared to WT), while levels of MCUR1 and MNCX were not altered (Figure 4B left and Table 2B). In addition, by cell-specific normalization of hMN qPCR data for all analyzed plasma membrane Ca2+ transporters to relative ChAT levels (and thus to respective hMN cell-sizes), 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). All other tested plasma membrane Ca2+ transporters (PMCA1-4 and NCX3) were not altered in hMNs from SOD1G93A mice compared to those of WT.


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

Mühling T, Duda J, Weishaupt JH, Ludolph AC, Liss B - Front Cell Neurosci (2014)

Elevated mRNA-levels of distinct Ca2+ transporters in individual hypoglossal motor neurons from SOD1G93A mice compared to WT. (A) Cell-specific RT-qPCR data for mitochondrial and plasma membrane Ca2+ transporters, derived from pools of 15 hMNs each from SOD1G93A mice and WT. Data are given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse brainstem tissue. (B) Data from (A), normalized to mitochondrially coded ND1 DNA-levels for mitochondrial Ca2+ transporters, and to ChAT cDNA levels for plasma membrane Ca2+ transporters. Significant differences according to Mann-Whitney-U-Tests are marked with (*), as defined in methods section. Note that data in (A,B) for the respective genes refer to two different axes, as indicated by the dashed line. (C) Right: Overview of concerted elevated Ca2+ transporter expression in individual hMN from SOD1G93A mice compared to WT (marked in red: MCU/MICU1, Letm1, UCP2, and NCX1). Left: These findings can provide a molecular basis for the recently described, selective functional, activity-dependent Ca2+ homeostasis deficit in hMN from endstage SOD1G93A mice. Insert shows stimulation protocol and resulting electrophysiological recordings (scale bar: 1 s), combined with calcium imaging (for details please see text and Fuchs et al., 2013).
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Figure 4: Elevated mRNA-levels of distinct Ca2+ transporters in individual hypoglossal motor neurons from SOD1G93A mice compared to WT. (A) Cell-specific RT-qPCR data for mitochondrial and plasma membrane Ca2+ transporters, derived from pools of 15 hMNs each from SOD1G93A mice and WT. Data are given as [pg/cell] in respect to a cDNA standard curve, generated from WT mouse brainstem tissue. (B) Data from (A), normalized to mitochondrially coded ND1 DNA-levels for mitochondrial Ca2+ transporters, and to ChAT cDNA levels for plasma membrane Ca2+ transporters. Significant differences according to Mann-Whitney-U-Tests are marked with (*), as defined in methods section. Note that data in (A,B) for the respective genes refer to two different axes, as indicated by the dashed line. (C) Right: Overview of concerted elevated Ca2+ transporter expression in individual hMN from SOD1G93A mice compared to WT (marked in red: MCU/MICU1, Letm1, UCP2, and NCX1). Left: These findings can provide a molecular basis for the recently described, selective functional, activity-dependent Ca2+ homeostasis deficit in hMN from endstage SOD1G93A mice. Insert shows stimulation protocol and resulting electrophysiological recordings (scale bar: 1 s), combined with calcium imaging (for details please see text and Fuchs et al., 2013).
Mentions: Next, via RT-qPCR, we analyzed qualitative mRNA expression of the main described potential mitochondrial and plasma membrane Ca2+ transporters (Figure 3A) in pools of 15 hMNs from WT and SOD1G93A mice. As illustrated in Figure 3B, mRNAs for all tested mitochondrial Ca2+ transporters (MCU/MICU1/MCUR1, Letm1, UCP2, MNCX) but UCP3, and for all plasma membrane Ca2+ transporters (PMCA1-4, NCX1, NCX3) but NCX2, were detected in ChAT and ND1 positive hMNs (n = 3 pools of 15 neurons each) from WT and SOD1G93A mice. Accordingly, we quantified mRNA-levels for all these mitochondrial and plasma membrane Ca2+ transporters via qPCR in hMNs from WT and endstage SOD1G93A mice. Results are given in Figure 4 and Table 2B. Without cell-specific normalization, we detected significantly higher mRNA levels of only MICU1 and UCP2 in hMNs from SOD1G93A mice (each about 1.7-fold higher compared to WT; Figure 4A left and Table 2B). All other Ca2+ transporter mRNA-levels were not significantly altered (although trends were observed in particular for MCU and Letm1, compare Figure 4A and Table 2B). Accordingly, cell-specific normalization of hMN qPCR data for mitochondrial Ca2+ transporters to mitochondrially coded ND1 DNA-levels (and thus to respective hMN mitochondrial genome numbers) revealed significantly higher mRNA levels of not only MICU1 and UCP2, but also of MCU and Letm1 in hMNs from SOD1G93A mice (each about 1.8-fold higher compared to WT), while levels of MCUR1 and MNCX were not altered (Figure 4B left and Table 2B). In addition, by cell-specific normalization of hMN qPCR data for all analyzed plasma membrane Ca2+ transporters to relative ChAT levels (and thus to respective hMN cell-sizes), 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). All other tested plasma membrane Ca2+ transporters (PMCA1-4 and NCX3) were not altered in hMNs from SOD1G93A mice compared to those of WT.

Bottom Line: This functional deficit was defined by a reduced hMN mitochondrial Ca(2+) uptake capacity and elevated Ca(2+) extrusion across the plasma membrane.These higher expression-levels of mitochondrial Ca(2+) transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification.Thus, pharmacological stimulation of Ca(2+) transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physiology, Institute of Applied Physiology, Ulm University Ulm, Germany.

ABSTRACT
Disturbances in Ca(2+) 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 Ca(2+) homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs) from a common ALS mouse model, the endstage superoxide dismutase SOD1(G93A) transgenic mouse, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca(2+) uptake capacity and elevated Ca(2+) extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca(2+) transporters in individual, choline-acetyltransferase (ChAT) positive hMNs from wildtype (WT) and endstage SOD1(G93A) 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 SOD1(G93A) mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca(2+) transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca(2+) transporters MCU/MICU1, Letm1, and UCP2 in remaining hMNs from endstage SOD1(G93A) mice. These higher expression-levels of mitochondrial Ca(2+) 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(+)/Ca(2+) exchanger NCX1 were also about 2-fold higher in hMNs from SOD1(G93A) mice. Thus, pharmacological stimulation of Ca(2+) transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS.

No MeSH data available.


Related in: MedlinePlus