Respiratory chain deficiency in aged spinal motor neurons

Abstract

Sarcopenia, muscle wasting, and strength decline with age, is an important cause of loss of mobility in the elderly individuals. The underlying mechanisms are uncertain but likely to involve defects of motor nerve, neuromuscular junction, and muscle. Loss of motor neurons with age and subsequent denervation of skeletal muscle has been recognized as one of the contributing factors. This study investigated aspects of mitochondrial biology in spinal motor neurons from elderly subjects. We found that protein components of complex I of mitochondrial respiratory chain were reduced or absent in a proportion of aged motor neurons-a phenomenon not observed in fetal tissue. Further investigation showed that complex I-deficient cells had reduced mitochondrial DNA content and smaller soma size. We propose that mitochondrial dysfunction in these motor neurons could lead to the cell loss and ultimately denervation of muscle fibers.

Keywords: Complex I; Mitochondria; Motor neuron; Sarcopenia; Spinal cord; mtDNA.

Fig. 1

Spinal cord tissue from 9 cases was analyzed for COX and/or SDH activity and protein expression of mitochondrial respiratory complexes. A representative image of respiratory-normal motor neurons in a ventral horn (brown) and a respiratory-deficient motor neuron (blue) (A). Expression of porin (mitochondrial outer membrane protein), complex II subunit 70 kDa (cII-70), and complex IV subunit I (COX-I) of mitochondrial respiratory chain were analyzed in 12 cases using IHC. Typical results are represented by 3 cases (case 7, 8, and 13) (B). Abbreviations: COX, cytochrome c oxidase; IHC, immunohistochemistry; SDH, succinate dehydrogenase. Scale bars measure 25μm (A) and 100μm (B).

Fig. 2

Complex I protein expression detected using antibodies to 2 subunits: 19 kDa (cI-19) and 20 kDa (cI-20) in paraffin-embedded lumbar spinal cord tissue sections from elderly individuals. Signal was developed using DAB (brown) or Vector SG (black), nuclei were counterstained with hematoxylin (blue). Representative images obtained from 4 cases are shown here (cases: 1, 5, 8, and 9). Arrows indicate motor neuron cell bodies: complex I-deficient (red), complex I-reduced (yellow), and complex I-normal (blue). Uneven staining reflects lipofuscin accumulation in motor neurons. Scale bars measure 50µm or 100μm.

Fig. 3

Complex I IHC in ventral and lateral areas of aged spinal cord obtained from case 8. cI-20 were visualized with Vector SG chromagen and cI-19 with DAB. All neuronal cell bodies in the lateral area express comparable levels of complex I proteins. Scale bars signify 100 μm. Abbreviation: IHC, immunohistochemistry. Scale bars signify 100μm.

Fig. 4

Protein expression levels of cI-19 and cI-20 (mean ± SD) from 12 elderly cases. Two to 3 sections per case were used for analysis and motor neurons were counted in both ventral horn areas. A scale developed based on visual scoring and densitometry allowed categorization of motor neurons into 3 groups: “+” complex I-normal (filled bars), “±“ complex I-reduced (checked bars), “−“ complex I-deficient (striped bars) (A). The proportion of motor neurons in each of the 3 groups in individual cases based on cI-20 (B) and cI-19 immunohistochemistry (C). Abbreviation: SD, standard deviation.

Fig. 5

Complex I deficiency in aged spinal cord sections assessed with dual immunofluorescence. cI-19 stained green (FITC) and subunit cII-70 stained red (TRITC), nuclei visualized with DAPI. Paraffin embedded spinal cord tissue section from an elderly subject (case 8) (10× magnification) (A). Single motor neurons from the same case (40× magnification) (B). Paraffin embedded brain section (cerebellum) from a patient with a POLG mutation, where severe complex I deficiency has been previously described (10× magnification) (C). Single complex I-deficient motor neurons from the same brain section (40× magnification) (D). Red arrows highlight complex I-deficient neurons (−), blue: complex I-normal (+) and yellow: complex I-reduced motor neurons (±). Abbreviation: POLG, polymerase γ. Scale bars measure: 100 μm (A), 20 μm (B, D), and 50 µm (C).

Fig. 6

Expression of complex I, II, and IV in fetal spinal cords. Motor neuron population depicted in 9wpc spinal cord section by ChAT labeling using Vector SG and expression of cI-20, COX-I, and cII-70 visualized using DAB (A). A different 9wpc fetal case dual immunolabelled for cI-20 and porin (B). In A scale bars measure 200μm (top panel) or 20 μm (bottom panel except for the image of ChAT: 50μm). In B scale bars signify 20 μm (top panel) or 10 μm (bottom panel).

Fig. 7

mtDNA deletion levels and mtDNA copy numbers in single motor neurons from 8 cases. Complex I-normal (+), complex I-reduced (±), and complex I-deficient (−) cells were compared. Each dot represents percentage of deletion in mtDNA from a single motor neuron (A). mtDNA copy numbers from complex I-normal (+) motor neurons were compared with complex I-reduced (±) and deficient (−) motor neurons. The difference was statistically significant (p-value = 0.021) (Wilcoxon rank-sum test). Each dot represents copy number reading for a single neuron normalized to an area unit (μm²) (B). Copy number of mtDNA in single motor neurons showing different complex I status from 8 elderly cases. Copy numbers of complex I-normal were compared with complex I-reduced and complex I-deficient motor neurons (C). Abbreviation: mtDNA, mitochondrial DNA.

Fig. 8

Size of individual motor neurons represented as area from 8 elderly cases. Each dot shows a measurement from a single cell. Differences in area were statistically significant with p-value < 0.0001 (A). A comparison between complex I-normal (+) and complex I-reduced (±) versus complex I-deficient (−) for individual cases (B).