Abstract
Age-related muscle weakness and loss of muscle mass (sarcopenia) is a universal problem in the elderly. Our previous studies indicate that alpha motor neurons (α-MNs) play a critical role in this process. The goal of the current study is to uncover changes in the aging spinal cord that contribute to loss of innervation and the downstream degenerative processes that occur in skeletal muscle. The number of α-MNs is decreased in the spinal cord of wildtype mice during aging, beginning in middle age and reaching a 41% loss by 27 months of age. There is evidence for age-related loss of myelin and mild inflammation, including astrocyte and microglia activation and an increase in levels of sICAM-1. We identified changes in metabolites consistent with compromised neuronal viability, such as reduced levels of N-acetyl-aspartate. Cleaved caspase-3 is more abundant in spinal cord from old mice, suggesting that apoptosis contributes to neuronal loss. RNA-seq analysis revealed changes in the expression of a number of genes in spinal cord from old mice, in particular genes encoding extracellular matrix components (ECM) and a 172-fold increase in MMP-12 expression. Furthermore, blood-spinal cord barrier (BSCB) permeability is increased in old mice, which may contribute to alterations in spinal cord homeostasis and exacerbate neuronal distress. Together, these data show for the first time that the spinal cord undergoes significant changes during aging, including progressive α-MNs loss that is associated with low-grade inflammation, apoptosis, changes in ECM, myelination, and vascular permeability.
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Funding
The study was supported by a grant from the National Institute on Aging (P01AG051442) awarded to HVR, and National Institutes of Health S10 grant 1S10OD023508 awarded to RT. KMP was supported by the Drs. Patricia H. and J. Donald Capra Fund Oklahoma Medical Research Foundation Predoctoral Scholarship. Dr. Van Remmen is the recipient of a Senior Research Career Scientist award (#1 IK6BX005234) from the Department of Veterans Afffairs.
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KMP designed research, conducted experiments, analyzed the results, and wrote the paper; SB contributed western blot and EM data; KS contributed to cytokine assay and the RNA-seq experiment; KSt contributed to the RNA-seq experiment; PP performed and analyzed isoprostanes assay; DS performed MRI; MZ and RG analyzed MRI data; SK, JL, RP, and BFM performed and analyzed data from the protein turnover assay; RT contributed to MRI experiment design, supervision, and interpretation; HVR supervised experiments and edited the paper.
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Fig. S1
Age-related α-motor neuron loss. Nuclear Fast Red staining of lumbar spinal cord sections. There is approximately a 39% decrease in α-MN number in old mice (females, 27 mo, n = 4, median and IQR = 7.542 and 2.455) comparing to young (6–7 mo, n = 4, median and IQR = 12.38 and 2.56; Mann-Whitney test, p = 0.0286); (PNG 40 kb)
Fig. S2
Blood vessel density does not decrease with age. A – A representative image of a cross section of lumbar spinal cord of a young (3 mo) and old (29 mo) mice stained for CD31 to visualize blood vessels (tiling of four 10x images, z-stack of 5, maximum intensity projection); B – quantification of blood vessel density a percentage of total SC area. There is no significant difference between young (n = 4, mean ± SD = 4.66 ± 1.79) and old mice (n = 7, mean ± SD = 5.42 ± 1.82; two-tailed unpaired t-test, t = 0.6720, p = 0.5185). (PNG 1017 kb)
Fig. S3
BSCB permeability to Gd-DTPA. MRI assessment of BSCB permeability; percentage change in the signal intensity pre- and post-Gd-DTPA injection in young (3–4 mo) and old (26–27 mo) mice measured in cervical (A), thoracic (B), and lumbar (C) spinal cord. (PNG 128 kb)
Supplementary Table 1
IPA analysis results on RNA-seq data from young and old spinal cord. Canonical pathways; Molecules; Diseases – main categories; Detailed diseases of functions; Predicted upstream regulators. (XLSX 81 kb)
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Piekarz, K.M., Bhaskaran, S., Sataranatarajan, K. et al. Molecular changes associated with spinal cord aging. GeroScience 42, 765–784 (2020). https://doi.org/10.1007/s11357-020-00172-6
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DOI: https://doi.org/10.1007/s11357-020-00172-6