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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

Scheme of UV-LMD and RT-qPCR protocol for quantitative expression analysis of Ca2+ transporters in individual hypoglossal motor neurons from endstage SOD1G93A and WT mice. (A) Nissl-stained sagittal (left) adult mouse brain section. The black bar indicates the zone (6–7 mm posterior to the Bregma) within the brainstem, where coronal sections (right) containing hypoglossal motor neurons (hMNs) (black box) were cut for UV-LMD. Scale bars: 1 mm. Pictures taken from Paxinos and Franklin (2001). (B) Upper: Overview of a WT (left) and endstage SOD1G93A mouse (right) coronal brainstem section after UV-LMD of 15 individual hMNs each. Scale bars: 250 μm. Inserts: photograph of the reaction tube cap for inspection of proper collection of all 15 neurons after UV-LMD, prior to cell lysis and reverse transcription (RT). Scale bars: 500 μm. Lower: individual hMNs before and after UV-LMD. Scale bars: 10 μm. (C) Workflow after UV-LMD. Left: genomic ND1 DNA-copy number of single hMNs was determined via qPCR with using genomic DNA as template, after genomic DNA isolation. ChAT and GFAP cDNA levels were determined for single hMNs after cell-lysis via individual RT-qPCR reactions with 50% of single cell cDNA each as templates. Right: For quantification of Ca2+ transporter expression-levels, cDNA derived from pools of 15 hMNs each were splitted, and 1/3 was used for marker gene expression profiling (either multiplex-nested PCR for ChAT, GFAP, and GAD65/67, or alternatively individual qPCRs for ChAT, GFAP, and ND1), and 2/3 was used for RT-qPCRs for quantification of Ca2+ transporter mRNA levels. For details, please see methods.
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Figure 1: Scheme of UV-LMD and RT-qPCR protocol for quantitative expression analysis of Ca2+ transporters in individual hypoglossal motor neurons from endstage SOD1G93A and WT mice. (A) Nissl-stained sagittal (left) adult mouse brain section. The black bar indicates the zone (6–7 mm posterior to the Bregma) within the brainstem, where coronal sections (right) containing hypoglossal motor neurons (hMNs) (black box) were cut for UV-LMD. Scale bars: 1 mm. Pictures taken from Paxinos and Franklin (2001). (B) Upper: Overview of a WT (left) and endstage SOD1G93A mouse (right) coronal brainstem section after UV-LMD of 15 individual hMNs each. Scale bars: 250 μm. Inserts: photograph of the reaction tube cap for inspection of proper collection of all 15 neurons after UV-LMD, prior to cell lysis and reverse transcription (RT). Scale bars: 500 μm. Lower: individual hMNs before and after UV-LMD. Scale bars: 10 μm. (C) Workflow after UV-LMD. Left: genomic ND1 DNA-copy number of single hMNs was determined via qPCR with using genomic DNA as template, after genomic DNA isolation. ChAT and GFAP cDNA levels were determined for single hMNs after cell-lysis via individual RT-qPCR reactions with 50% of single cell cDNA each as templates. Right: For quantification of Ca2+ transporter expression-levels, cDNA derived from pools of 15 hMNs each were splitted, and 1/3 was used for marker gene expression profiling (either multiplex-nested PCR for ChAT, GFAP, and GAD65/67, or alternatively individual qPCRs for ChAT, GFAP, and ND1), and 2/3 was used for RT-qPCRs for quantification of Ca2+ transporter mRNA levels. For details, please see methods.

Mentions: Carried out essentially as described (Fuchs et al., 2013; Schlaudraff et al., 2014). Briefly, SOD1G93A and WT mice were deeply anesthetized with isoflurane (Abbott, Wiesbaden, Germany) and decapitated. Coronal tissue blocks containing hypoglossal nuclei were separated. The blocks were mounted on a specimen disk and immediately frozen by insertion into the snap-freeze holder (−35°C) of a cryostat (Leica CM 1850). Twelve μm serial coronal brainstem sections (for exact location see Figure 1A) were cut using a microtome blade (type R35, Feather, Osaka, Japan), and mounted on 2 mm PEN-membrane slides (Microdissect, Herborn, Germany), fixed with an ascending ethanol series, stained with cresyl violet, dried and stored at −80°C. UV-LMD of individual hMNs was performed using a Leica LMD7000 setup. 10 pools of 15 hMNs each were laser microdissected from each endstage SOD1G93A and age-matched WT mouse. After cell-lysis and reverse transcription (RT) with random hexamer primers, cDNA was ethanol precipitated as described (Liss, 2002), resolved in 17 μl molecular biology grade water and stored at −20°C until PCR amplification. Note that hMNs of SOD1G93A mice were about 5% larger than hMNs of WT control mice, according to area-quantifications after UV-LMD.


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)

Scheme of UV-LMD and RT-qPCR protocol for quantitative expression analysis of Ca2+ transporters in individual hypoglossal motor neurons from endstage SOD1G93A and WT mice. (A) Nissl-stained sagittal (left) adult mouse brain section. The black bar indicates the zone (6–7 mm posterior to the Bregma) within the brainstem, where coronal sections (right) containing hypoglossal motor neurons (hMNs) (black box) were cut for UV-LMD. Scale bars: 1 mm. Pictures taken from Paxinos and Franklin (2001). (B) Upper: Overview of a WT (left) and endstage SOD1G93A mouse (right) coronal brainstem section after UV-LMD of 15 individual hMNs each. Scale bars: 250 μm. Inserts: photograph of the reaction tube cap for inspection of proper collection of all 15 neurons after UV-LMD, prior to cell lysis and reverse transcription (RT). Scale bars: 500 μm. Lower: individual hMNs before and after UV-LMD. Scale bars: 10 μm. (C) Workflow after UV-LMD. Left: genomic ND1 DNA-copy number of single hMNs was determined via qPCR with using genomic DNA as template, after genomic DNA isolation. ChAT and GFAP cDNA levels were determined for single hMNs after cell-lysis via individual RT-qPCR reactions with 50% of single cell cDNA each as templates. Right: For quantification of Ca2+ transporter expression-levels, cDNA derived from pools of 15 hMNs each were splitted, and 1/3 was used for marker gene expression profiling (either multiplex-nested PCR for ChAT, GFAP, and GAD65/67, or alternatively individual qPCRs for ChAT, GFAP, and ND1), and 2/3 was used for RT-qPCRs for quantification of Ca2+ transporter mRNA levels. For details, please see methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4231948&req=5

Figure 1: Scheme of UV-LMD and RT-qPCR protocol for quantitative expression analysis of Ca2+ transporters in individual hypoglossal motor neurons from endstage SOD1G93A and WT mice. (A) Nissl-stained sagittal (left) adult mouse brain section. The black bar indicates the zone (6–7 mm posterior to the Bregma) within the brainstem, where coronal sections (right) containing hypoglossal motor neurons (hMNs) (black box) were cut for UV-LMD. Scale bars: 1 mm. Pictures taken from Paxinos and Franklin (2001). (B) Upper: Overview of a WT (left) and endstage SOD1G93A mouse (right) coronal brainstem section after UV-LMD of 15 individual hMNs each. Scale bars: 250 μm. Inserts: photograph of the reaction tube cap for inspection of proper collection of all 15 neurons after UV-LMD, prior to cell lysis and reverse transcription (RT). Scale bars: 500 μm. Lower: individual hMNs before and after UV-LMD. Scale bars: 10 μm. (C) Workflow after UV-LMD. Left: genomic ND1 DNA-copy number of single hMNs was determined via qPCR with using genomic DNA as template, after genomic DNA isolation. ChAT and GFAP cDNA levels were determined for single hMNs after cell-lysis via individual RT-qPCR reactions with 50% of single cell cDNA each as templates. Right: For quantification of Ca2+ transporter expression-levels, cDNA derived from pools of 15 hMNs each were splitted, and 1/3 was used for marker gene expression profiling (either multiplex-nested PCR for ChAT, GFAP, and GAD65/67, or alternatively individual qPCRs for ChAT, GFAP, and ND1), and 2/3 was used for RT-qPCRs for quantification of Ca2+ transporter mRNA levels. For details, please see methods.
Mentions: Carried out essentially as described (Fuchs et al., 2013; Schlaudraff et al., 2014). Briefly, SOD1G93A and WT mice were deeply anesthetized with isoflurane (Abbott, Wiesbaden, Germany) and decapitated. Coronal tissue blocks containing hypoglossal nuclei were separated. The blocks were mounted on a specimen disk and immediately frozen by insertion into the snap-freeze holder (−35°C) of a cryostat (Leica CM 1850). Twelve μm serial coronal brainstem sections (for exact location see Figure 1A) were cut using a microtome blade (type R35, Feather, Osaka, Japan), and mounted on 2 mm PEN-membrane slides (Microdissect, Herborn, Germany), fixed with an ascending ethanol series, stained with cresyl violet, dried and stored at −80°C. UV-LMD of individual hMNs was performed using a Leica LMD7000 setup. 10 pools of 15 hMNs each were laser microdissected from each endstage SOD1G93A and age-matched WT mouse. After cell-lysis and reverse transcription (RT) with random hexamer primers, cDNA was ethanol precipitated as described (Liss, 2002), resolved in 17 μl molecular biology grade water and stored at −20°C until PCR amplification. Note that hMNs of SOD1G93A mice were about 5% larger than hMNs of WT control mice, according to area-quantifications after UV-LMD.

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