Expression analysis of mitochondrial and plasmalemmal calcium transporters in individual hypoglossal motor neurons of endstage superoxide dismutase 1 transgenic mice
Ludolph, Albert C.
InstitutionenInstitut für Angewandte Physiologie
UKU. Klinik für Neurologie
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting particularly cholinergic motor neurons (MN) of the motor cortex, the brainstem and the spinal cord, and leading to paralysis and atrophy of the innervated muscles. Most patients die within two to four years after onset of the first symptoms. Glutamate excitotoxicity, associated disturbances in calcium (Ca2+) homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial as well as sporadic forms of ALS. However, the distinct molecular pathology, in particular the cause for the differential vulnerability of different motor neuron subtypes to disease triggers, remains unclear. Recently, in a common ALS model, the endstage superoxide dismutase 1 (SOD1G93A) transgenic mouse, an activity-dependent, complex Ca2+ clearance deficit, present selectively in highly vulnerable motor neurons of the hypoglossal nucleus (hMNs) was described by our group. This functional deficit was defined by a reduced mitochondrial Ca2+ uptake and an elevated Ca2+ extrusion across the plasma membrane, selectively in hMNs of endstage SOD1G93A mice, while Ca2+ clearance via the endoplasmic reticulum (ER) remained preserved. To address the underlying molecular mechanisms, I (1) transferred an established combined ultraviolett laser microdissection (UV-LMD) and realtime quantitative polymerase chain reaction (RT-qPCR) protocol for cell specific messenger RNA (mRNA) quantification of individual neurons from mouse midbrain sections to a respective mouse brainstem preparation and hypoglossal motor neurons from adult wildtype (WT) and SOD1G93A mice and (2) established suitable primers to analyze expression levels of respective mitochondrial (mitochondrial calcium uniporter (MCU), mitochondrial calcium uptake 1 (MICU1), mitochondrial calcium uniporter regulator 1 (MCUR1), leucine zipper EF-hand containing transmembrane protein 1 (LETM1), uncoupling proteins 2 and 3 (UCP2, UCP3), mitochondrial sodium-calcium exchanger (MNCX)) and plasma membrane Ca2+ transporters (sodium-calcium exchanger 1-3 (NCX1-3), plasma membrane calcium ATPase (PMCA 1-4)) in individual, choline-acetyltransferase (ChAT) positive hMNs from wildtype and endstage SOD1G93A mice. In addition (3), to further stratify single cell UV-LMD RT-qPCR data derived from mouse hMNs, suitable normalization strategies were tested (normalization to cell size, as well as to the cytosolic marker gene ChAT and the mitochochondrial marker gene NADH dehydrogenase 1 (ND1). And finally (4), I compared with this optimized protocol mRNA expression levels of mitochondrial and plasma membrane Ca2+ transporters in individual hMNs from endstage SOD1G93A and WT mice. With this optimized method I found (1) that ChAT as well as ND1 mRNA levels are similar in hMNs from SOD1G93A and WT mice. This indicates (i) that neither neurotransmitter synthesis nor number of mitochondria/mitochondrial genoms is altered in hMNs of endstage SOD1 mice, and (ii) that ChAT as well as ND1 mRNA levels are suitable for normalization of RT-qPCR data derived from mouse hMNs. I also found that (2) all tested Ca2+ transporters but UCP3 and NCX2 were expressed in mouse hMNs. Furthermore (3), with cell specific normalization of RT-qPCR data for mitochondrial Ca2+ transporters to ND1 and plasmalemmal Ca2+ transporters to ChAT, I detected about 2-fold higher mRNA 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. In addition (4), normalized mRNA-levels for the plasma membrane Na+/Ca2+ exchanger NCX1 were also about 2-fold higher in hMNs from SOD1G93A mice. This concerted up-regulation of distinct mitochondrial and plasma membrane Ca2+ transporters with different locations as well as transport kinetics might serve as a complex compensatory response to the disease trigger and to the altered Ca2+ homeostasis, and protect surviving hMNs in SOD1G93A mice from degeneration. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS. However further studies a necessary to address cell specific causes and consequences of altered Ca2+ transporter activity in motor neurons – in mouse models and, most important, in human motor neurons from ALS patients and controls.
Erstellung / Fertigstellung
Normierte SchlagwörterMyatrophische Lateralsklerose [GND]
Nervus hypoglossus [GND]
Real time quantitative PCR [GND]
Amyotrophic lateral sclerosis [MeSH]
Hypoglossal nerve [MeSH]
Superoxide dismutase [MeSH]
Motor neurons [MeSH]
Laser capture microdissection [MeSH]
Polymerase chain reaction [MeSH]
Mice, transgenic [MeSH]