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. Author manuscript; available in PMC: 2021 Mar 17.
Published in final edited form as: Exp Gerontol. 2020 Dec 5;144:111193. doi: 10.1016/j.exger.2020.111193

Age-related impairment of autophagy in cervical motor neurons

Miguel Pareja-Cajiao a, Heather M Gransee a, Jessica M Stowe a, Sabhya Rana b, Gary C Sieck a,b, Carlos B Mantilla a,b,*
PMCID: PMC7968728  NIHMSID: NIHMS1677332  PMID: 33290859

Abstract

Neuromuscular dysfunction is common in old age. Damaged cytoplasmic structures aggregate with aging, especially in post-mitotic cells like motor neurons. Autophagy is a ubiquitous cell process that aids in the clearance of damaged aggregates. Accordingly, we hypothesized that autophagy is impaired in old age, contributing to neuromuscular dysfunction via an effect in motor neurons. Autophagy flux may be impaired as a result of deficits in the initiation, elongation or degradation phases. Changes in the expression levels of core proteins necessary for each of the autophagy phases were evaluated by Western blotting in the cervical spinal cord (segments C2-C6 corresponding to the phrenic motor pool) of adult male and female mice at 6-, 18-, and 24-months of age (reflecting 100%, 90% and 75% survival, respectively). There was no evidence of an effect of age on the expression of the autophagy markers Beclin-1 (Becn-1; initiation), ATG7 and ATG5/12 complex (elongation) or LC3 (elongation/degradation). Reduced p62 expression (a marker of degradation) was evident in the cervical spinal cord of adult mice at 18-months compared to 24-months. Accordingly, expression of LC3 and p62 in motor neurons was analyzed using immunofluorescence and confocal microscopy in separate animals. LC3 and p62 immunoreactivity was evident in the gray matter with minimal expression in the white matter across all age groups. A mixed linear model with animal as a random effect was used to compare relative LC3 and p62 expression in motor neurons to gray matter across age groups. Expression of both LC3 and p62 was higher in choline acetyl transferase (ChAT)-positive motor neurons (~2–3 fold vs. gray matter). Across age groups, there were differences in the relative expression of LC3 (F2,12 = 7.59, p < 0.01) and p62 (F2,12 = 8.00, p < 0.01) in cervical motor neurons. LC3 expression in motor neurons increased ~20% by 24-months of age in both male and female mice. p62 expression in motor neurons increased ~70% by 18-months compared to 6-months with no further changes by 24-months of age in male mice. p62 expression did not change across age groups in female mice, and was ~20% higher than in males. Our findings highlight important changes in autophagy pathways that likely contribute to the development of aging-related neuromuscular dysfunction in mice. At 18-months of age, increased autophagosome clearance (reduced p62 expression) appears to be a global effect not restricted to motor neurons. By 24-months of age, increased expression of LC3 and p62 indicates impaired autophagy with autophagosome accumulation in cervical motor neurons.

Keywords: Autophagy, Aging, Motor neuron, Spinal cord, Neuromuscular dysfunction

1. Introduction

Aging is a series of processes characterized by alterations in multiple tissues that lead to physiological dysfunction and increased vulnerability to death. Ventilatory impairments increase in old age and respiratory complications are an important cause of death (Fein and Niederman, 1994; Heron, 2017; Houston et al., 1997). Aging effects on the phrenic motor system comprise increased neuromuscular transmission failure (Greising et al., 2015a), increased denervation of diaphragm muscle neuromuscular junctions (Greising et al., 2015d), diaphragm muscle sarcopenia (Greising et al., 2013) and loss of phrenic motor neurons (Fogarty et al., 2018). The mechanism(s) causing aging-related neuromuscular dysfunction in the phrenic motor system are not completely understood. Accumulation of damaged cytoplasmic structures is a hallmark of aging and causes cellular dysfunction (Lopez-Otin et al., 2013; Powers et al., 2009). Cellular mechanisms that cope with the accumulation of damaged cytoplasmic structures, including autophagy (Frake et al., 2015), are impaired with aging and may thus contribute to dysfunction of the neuromuscular system (Gonzalez Porras et al., 2018; Jiao and Demontis, 2017).

Autophagy is a tightly regulated, multistep process (Perluigi et al., 2015; Ravikumar et al., 2010; Yang and Klionsky, 2010) that clears damaged cytoplasmic structures (Fig. 1), and comprises the sequestration of damaged cytoplasmic structures in a double-membrane vacuole called autophagosome, which is then delivered to the lysosome for degradation (Farre and Subramani, 2016; Nixon, 2013). Aging-related impairment of autophagy in motor neurons and the resulting reduced clearance of damaged cytoplasmic structures, may contribute to altered axonal trafficking (Ashrafi et al., 2014), denervation of muscle fibers (Greising et al., 2015b, 2015d), subsequent muscle atrophy and force loss (sarcopenia) (Greising et al., 2013), and motor neuron death (Fogarty et al., 2018; Liang and Sigrist, 2018; Mizushima et al., 2008). Accordingly, we hypothesize that autophagy is impaired with aging in cervical motor neurons, and used complementary techniques to examine markers of autophagy during a period of vulnerability to aging effects.

Fig. 1.

Fig. 1.

Fig. 1.

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A) Autophagy comprises a series of evolutionarily conserved phases by which cells target damaged intracellular components for degradation in lysosomes. During the initiation phase, damaged organelles trigger activation of the BCL-2-interacting protein Becn-1 (Becn1) whereas proteins targeted for degradation become ubiquitinated. During the elongation phase of autophagy, recruitment of ATG7 and the ATG5/12 complex results in the lipidation of LC3 (reflected in the conversion of LC3-I to LC3-II) and its incorporation into nascent autophagic vacuole membranes. Damaged organelles and ubiquitinated proteins are also incorporated into autophagic vacuoles via the specific autophagy receptor protein p62. Once the mature autophagic vacuole membrane envelops its cargo, it is then delivered to the lysosome, where all its contents are degraded, including p62. Accordingly, degradation of p62 is used to monitor the degradation phase. Figure modified from (Gonzalez Porras et al., 2018). B) Higher expression of Becn1, ATG7, the ATG5/12 complex and LC3 reflect increased production of autophagosomes, whereas an accumulation of p62 represents lack of corresponding autophagosome degradation. The figure represents the conceptual framework for the interpretation of changes in the expression of autophagy markers as a result of aging or other conditions.

2. Materials and methods

2.1. Animals

Experiments used adult male and female C57BL/6 mice at 6-, 18- and 24-months of age (reflecting 100%, 90% and 75% survival, respectively). Mice (n = 42) were housed at the Mayo Clinic, group caged by sex and age, and maintained with a 12-h light cycle with free access to food and water. All protocols were approved by the Institutional Animal Care and Use Committee, in compliance with the National Institutes of Health guidelines. At the terminal experiments, mice were weighed, anesthetized with intraperitoneal fentanyl (0.3 mg/kg), diazepam (5 mg/kg) and droperidol (15 mg/kg) and euthanized by exsanguination.

2.2. Western blotting

Autophagy was first evaluated by measuring expression of key markers for each phase of autophagy with Western blotting in a subset of male and female mice (n = 24). Segments C2-C6 of the cervical spinal cord were harvested, frozen in isopropane cooled in liquid N2 and stored at −80 °C. Proteins were extracted using ice-cold 1× RIPA buffer (#9806S; Cell Signaling, Danvers, MA) in presence of protease inhibitors (complete®; Roche Diagnostics, Mannheim, Germany). Protein concentration was determined by Bradford assay (DC™ Protein Assay #500-0115; Bio-Rad, Hercules, CA) run on SpectraMax Plus 384 (Molecular Devices, San Jose, CA) microplate reader. Equal amounts of protein (75 μg) per lane separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (10–20% gradient gel, Bio-Rad) and then transferred onto polyvinylidene difluoride membranes (Bio-Rad). All three age groups were included in each gel. Membranes were blocked in 1× Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% bovine serum albumin or 5% non-fat dry milk, and then incubated in primary antibody: anti-Becn-1 (#3495, 1:1000; Cell Signaling); anti-ATG7 (#8558, 1:1000; Cell Signaling) anti-ATG5 (#NB110-53818, 1:1000; Novus, Littleton, CO); anti-LC3B (#NB100-2220, 1:1000; Novus), anti-p62 (#ab56416, 1:1000; Abcam, Cambridge, MA), and anti-GAPDH (#g9545, 1:7500; Sigma, St Louis, MO). After several washes, an appropriate peroxidase-conjugated secondary antibody (goat IgG, #111-035-003/#115-035-003; Jackson Immuno, West Grove, PA) was applied. Peroxidase activity was visualized using ECL Western Blotting Detection kit (SuperSignal™, Dura ECL, Thermo Fisher Scientific, Waltham, MA). Signal intensity was detected on Image Station using the Kodak Molecular Imaging Software. For quantitative analyses, Becn-1, ATG7, ATG5/12, LC3B and p62 were corrected for the corresponding GAPDH signal. For the ATG5 marker, only the ATG5/12 complex band (55 kDa) was analyzed (Otomo et al., 2013). Within each membrane, data were summarized relative to the average data for the 6 month-old group and quantified as relative expression (fold-change).

2.3. Immunofluorescence

A subset of animals, including adult male and female C57BL/6 mice at 6-, 18- and 24-months of age (n = 18) was used for immunofluorescence analyses. Briefly, after euthanasia animals were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered solution (pH 7.4) prior to tissue collection. The cervical spinal cord was resected from C1 to C6, and kept in 4% paraformaldehyde overnight and transferred to 30% sucrose in 0.1 M phosphate-buffered solution (pH 7.4) at 4 °C. The whole resected spinal cords were assembled such that the cervical spinal cords from three animals could be sectioned simultaneously in the transverse plane and thaw-mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), with each slide containing up to 5 sections per animal. This stereological approach was used to generate ten equally spaced sets representing the entirety of the cervical spinal cord. Slides were kept at −80 °C until further processing.

A single set of ~35 sections per animal (cut at 20 μm thick and 200 μm apart) was selected for immunofluorescence using a random number generator. For the selected set, sections were washed in TBS to remove excess OCT followed by antigen retrieval in 10 mM tri-sodium citrate containing 0.05% Tween 20 (pH 6.0) at 80 °C. After blocking in 10% donkey serum in 0.3% Triton-TBS, sections were incubated with primary antibodies (all in 5% donkey serum in 0.3% Triton-TBS) for LC3B (NB100-2220, rabbit; 1:200; Novus), p62 (GPg2-C, guinea pig; 1:100; Progen, Wayne, PA) and choline acetyl transferase (ChAT - AB144P, goat; 1:200; Millipore, Burlington, MA) at 4 °C. After wash, sections were incubated in Alexa Fluor-conjugated secondary antibodies from Jackson Immuno (Alexa Fluor 647 donkey-anti-rabbit [711-605-152], Alexa Fluor 594 donkey-anti-guinea pig [706-585-148], and Alexa Fluor 488 donkey-anti-goat [705-545-147]). Sections were treated with Pro-Long™ Gold containing DAPI (Thermo Fischer), cover-slipped and allowed to cure before imaging.

2.4. Antibody validation

Autophagy is a ubiquitous process and the markers evaluated are expressed in all tissues. Thus, the presence of a single band at the expected and reported size in a Western blot was used as a positive control. This step was performed using several tissues, including cerebellum and lumbar spinal cord, in addition to the cervical spinal cord. When multiple bands were evidenced in a blot (LC3B and ATG5 antibodies), the available blocking peptides for LC3B (NB100-2220PEP, 1:100; Novus) and ATG5 (NB110-53818PEP, 1:100; Novus) were used to detect the specific bands of interest. For immunofluorescence, the presence of intracellular immunofluorescence in ChAT-positive motor neurons in the cervical spinal cord and tubular epithelium in the kidney was used as a positive control. Separate sections were also incubated in 5% donkey serum in 0.3% Triton-TBS without the primary antibodies as negative controls.

2.5. Confocal microscopy

Immunofluorescent labeling was detected using an Olympus Fluo-View FV1200 laser scanning confocal system (Olympus America Inc., Melville, NY) with four diode lasers (405, 488, 559 and 635 nm) and four fluorescence detectors mounted on an Olympus BX61 microscope. This confocal system was used to optically section multi-labeled fluorescent samples using the Fluoview software. All images for a single animal were taken using the same laser settings. Individual images obtained using the panoramic 10× objective (NA 0.3) were stitched to evaluate gray matter anatomy and determine spinal cord segment (Fig. 2A) using a standard atlas (Sengul et al., 2012); no intensity analyses were performed using this lower magnification image. Sections determined to be part of segments C3-C5 were imaged using an oilimmersion 20× objective (NA 0.8). Image stacks (2 μm step size) comprising ChAT-positive neurons were obtained for each side of the ventral horn (Fig. 2C).

Fig. 2.

Fig. 2.

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Methods for immunohistochemical measurements of LC3B and p62 expression in motor neurons. Low magnification, stitched image (A) of the spinal cord in cross-section, used to identify the spinal segment by comparison to standard atlases. Multicolor-labeled fluorescence image shows LC3B and p62 in the ventral horn of the cervical spinal cord at C4 predominately in the gray matter regions. Insert represents area used to obtain high magnification image in (B) and (C). LC3B and p62 fluorescence (B) was measured in motor neurons identified by ChAT immunoreactivity (C) using manually generated regions of interest (ROIs) in the maximum projection images. See Methods for details. ChAT, Choline-Acetyl Transferase. Scale bar in 200 μm (A) and 100 μm (B and C).

2.6. Image analysis

Confocal image stacks were processed using Metamorph (Molecular Devices, San Jose, CA) and NIS-Elements (Nikon, Melville, NY). Images where the tissue displayed extensive damage or folds were not included for analyses. Regions of interest (ROIs) were drawn as follows: three ROIs of constant shape and area in both the gray matter and white matter, excluding sites that showed micro-tears in the tissue (Fig. 2B). In the ChAT channel, motor neurons were identified as large ChAT-immunoreactive cells with a cross-sectional area of more than 200 μm2 and surrounded by pericellular ChAT-immunoreactive boutons. A ROI was manually drawn around each motor neuron displaying mid-nuclear section in the Z-stack (Fig. 2C).

Both fluorescence channels (LC3B and p62) were analyzed separately after obtaining a maximum intensity projection image for each section. For each spinal cord section within the C3-C5 spinal cord, mean fluorescence intensity was determined for every ROI (motor neuron, gray and white matter). The average fluorescence intensity for the three white matter ROIs was used as background and subtracted from all other ROIs. The ratio of the mean fluorescence intensity of each motor neuron to the average of gray matter ROIs in that section was then determined. Each motor neuron was identified as a putative phrenic motor neuron or as belonging to non-phrenic motor neuron pools based on its location within the gray matter, compared to a standard mouse spinal cord atlas (Sengul et al., 2012). The relative fluorescence intensity for LC3B and p62 (segregated by motor neuron pool) was then averaged in every section for statistical analyses. These data were averaged for each animal for presentation. In additional analyses, the relative fluorescence intensity of LC3B was plotted vs. the relative fluorescence intensity of p62 in each motor neuron, segregated by motor neuron pool. In these analyses per motor neuron, Z-scores of the relative fluorescence intensity were calculated from the overall mean and SD for all animals.

2.7. Statistical analyses

All statistical evaluations were performed using standard statistical software (JMP 11, SAS Institute Inc., Cary, NY). Aging effects on body weight were compared using age, sex, and their interaction in a two-way ANOVA. Protein expression determined by Western blotting (Becn-1, ATG7, ATG5/12 complex, LC3B-I, LC3B-II, LC3B-II/I and p62) was compared using a one-way ANOVA with age (6-, 18- and 24-months of age). Relative motor neuron fluorescence intensity for LC3B and p62 in each section was compared in a mixed linear model using age, motor neuron pool (phrenic vs. non-phrenic) and their interaction as fixed effects, and with animal as a random effect. When appropriate, post hoc analyses were conducted using Tukey-Kramer HSD test. Prior studies did not suggest sex-based differences in aging effects on the phrenic motor system (Greising et al., 2017; Khurram et al., 2018), but all data were plotted to allow visual inspection of possible sex differences. Relative fluorescence intensity measurements suggested a possible sex effect and thus in an additional analysis per motor neuron, a mixed linear model with age, sex and their interaction was conducted for the Z-score adjusted LC3B and p62 fluorescence. In all cases, statistical significance was established at p < 0.05. Data are presented in arbitrary units, with Western blot data compared to the 6-month-old value for each blot, and the relative fluorescence intensity data compared to the background-subtracted gray matter value for each section. All the experimental data in this text are presented as mean ± 95% confidence interval (CI), unless otherwise specified.

3. Results

3.1. Aging effects on body weight

A total of 42 mice equally balanced by sex were used in the experiments (total: 24 for Western blotting and 18 for immunofluorescence experiments). There was a significant effect on body weight of both age (F2,30 = 6.2; p = 0.005) and sex (F1,30 = 37.7; p < 0.001), but no interaction (F2,30 = 0.9, p = 0.4). On average, males weighed 32.9 ± 0.7 g and females weighed 27.4 ± 0.7 g. The average weight was 28.2 ±1.3 g, 30.5 ± 1.0 g and 31.9 ± 1.0 g for the 6-, 18- and 24-month old groups. Male mice displayed significantly greater weight than females of the same age group for the 6- and 18-month old groups, but this difference was no longer significant by 24 months of age.

3.2. Aging has no major effect on the initiation or elongation phases of autophagy

There was no evidence of an aging effect on the expression of Becn-1, ATG7 and the ATG5/12 complex in cervical spinal cord homogenates of mice at 6-, 18- and 24-months of age (Fig. 3). Activation of Becn-1 characterizes the initiation phase and its expression levels are widely used to monitor autophagy (Gonzalez Porras et al., 2018; Klionsky et al., 2016). There was no effect on Becn-1 expression of age (F2,21 = 0.49, p = 0.62). The elongation phase comprises the recruitment of ATG7 and the ATG5/12 complex to assemble a double-membrane lipid vacuole via LC3 lipidation (Kaur and Debnath, 2015). There was no effect on ATG7 expression of age (F2,21 = 0.43, p = 0.66). There was also no effect on ATG5/12 complex expression of age (F2,21 = 0.06, p = 0.94).

Fig. 3.

Fig. 3.

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Expression of the initiation and elongation autophagy markers Becn-1 (A), ATG 7 (B) and the ATG5/12 complex (C) as determined using Western blotting in the cervical spinal cord of mice across age groups. Bar graphs show mean ± 95% CI of individual measurements per animal (top) and corresponding representative blots (bottom). There were no significant differences in the protein expression levels of these autophagy markers across age groups.

3.3. Phagophore markers accumulate in motor neurons with aging

There was no evidence of an aging effect on the expression of LC3-I, LC3-II or the LC3-II/I ratios in cervical spinal cord homogenates of mice at 6-, 18- and 24-months of age (Fig. 4A-D). The lipidation of the microtubule-associated protein light chain 3 (LC3) marks the maturation of autophagosomes (Klionsky et al., 2016). There was no effect on LC3-I expression of age (F2,21 = 2.56; p = 0.10). There was no effect on LC3-II expression of age (F2,21 = 0.48; p = 0.62). With the conversion of LC3-I to LC3-II, the elongating membrane engulfs the tagged cargos inside autophagosomes (Xie and Klionsky, 2007; Yang and Klionsky, 2009). Accordingly, the LC3-II/I ratio can be used to monitor autophagy flux. There was no effect on the LC3-II/I ratio of age (F2,21 = 0.67; p = 0.51).

Fig. 4.

Fig. 4.

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Expression of the autophagosome marker LC3 in the cervical spinal cord and motor neurons of mice across age groups. Representative blot (A). Bar graphs show mean ± 95% CI of individual protein expression measurements of LC3-I (B), LC3-II (C) and the LC3-II/I ratio (D) per animal. There were no age-related differences in the protein expression levels of these markers or in the LC3-II/I ratio in cervical spinal cord of mice across age groups. Representative high magnification image of LC3 and ChAT immunofluorescence in motor neurons of mice at 6-, 18-, and 24-months of age (E). Bar graphs show mean ± 95% CI of LC3 expression in motor neurons, averaged per section and animal (for details, see methods section) in putative phrenic motor neurons (F) and non-phrenic motor neurons (G). LC3B expression in putative phrenic motor neurons at 24-months of age was ~25% higher than both the 6- and 18-month old groups. There were no age-related differences in LC3B expression at non-phrenic motor neurons. Scale bar: 100 μm; *, statistically significant difference between groups, post hoc Tukey-Kramer HSD, p < 0.05.

In order to determine any age-related changes in LC3 expression in motor neurons, additional experiments measured the relative fluorescence intensity of LC3B in ChAT-positive motor neurons in segments C3–5 of the cervical spinal cord (Fig. 4E-G). Across age groups, motor neurons evidenced ~2-fold greater intensity of LC3B relative to the gray matter. There was evidence of a significant effect on LC3 expression of age (F2,15 = 4.50; p = 0.03) and motor neuron pool (putative phrenic motor neurons vs. other cervical motor neurons; F1,325 = 6.18; p = 0.01), but no age*motor neuron pool interaction (F2,325 = 2.91; p = 0.06). Overall, LC3B expression increased by ~25% at 24-months of age when compared to both the 6- and 18-month old groups and was ~5% greater in putative phrenic motor neurons relative to non-phrenic motor neurons. Putative phrenic motor neurons at 24-months of age displayed a ~30% increase in relative LC3B fluorescence intensity compared to the 6-monh old group. There was no effect of age on the non-phrenic cervical motor neurons.

3.4. Autophagosome degradation is impaired with aging

We found evidence of an aging effect on the expression of p62 in cervical spinal cord homogenates and motor neurons of mice at 6-, 18- and 24-months of age (Fig. 5). The degradation phase is characterized by the recycling of all the contents inside the autophagosomes, including p62 (Katsuragi et al., 2015; Pankiv et al., 2007; Sanchez-Martin and Komatsu, 2018). An increase in autophagy augments p62 clearance, whereas an impairment of autophagy causes p62 to accumulate (Cha-Molstad et al., 2017). There was an effect on the expression of p62 of age (F2,21 = 5.95; p < 0.01). The expression of p62 in cervical spinal cord homogenates increased by ~2-fold in 24-month old mice when compared to 18-months, and was not different between the 6-month old group and the 18- or the 24-month old groups.

Fig. 5.

Fig. 5.

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Representative blot showing protein expression of p62 (A). Bar graphs show mean ± 95% CI of p62 expression in motor neurons. p62 expression in cervical spinal cord of mice was ~2-fold higher at 24 months of age compared to the 18-month old group (B). Representative high magnification image of p62 and ChAT immunofluorescence in motor neurons at 6-, 18- and 24-months of age (C) same subset of motor neurons as displayed in Fig. 4E. Bar graphs show mean ± 95% CI of p62 expression in motor neurons, averaged per section and animal (for details, see methods section) in putative phrenic motor neurons (D) and non-phrenic motor neurons (E). Phrenic motor neurons at 18- and 24-months of age displayed ~35% higher p62 expression than the 6-month old group. Non-phrenic motor neurons at 24-months of age displayed ~35% higher p62 expression than the 6- but not the 18-month old group. Scale bar: 100 μm; *, statistically significant difference between groups in brackets, post hoc Tukey-Kramer HSD, p < 0.05.

In order to determine any age-related changes in p62 expression in motor neurons, further experiments measured the relative fluorescence intensity of p62 in ChAT-positive motor neurons (Fig. 5C-E). Across age groups, motor neurons displayed ~2–3-fold greater intensity of p62 relative to the remainder of the background-subtracted gray matter. There was an effect on p62 expression of age (F2,15 = 4.05; p = 0.04) and motor neuron pool (F1,325 = 6.04; p = 0.01), but no age*pool interaction (F2,326 = 0.34; p = 0.72). Overall, p62 expression increased by ~35% by 24-months of age when compared to the 6-month old group, and was ~5% higher in putative phrenic motor neurons than non-phrenic motor neurons in the cervical spinal cord.

3.5. Correlation between aging-related changes in LC3B and p62 at individual motor neurons

Visual examination of the results of motor neuron expression for LC3 (Fig. 4F-G) and p62 (Fig. 5D-E) showed clustering by sex. Accordingly, we also examined the correlation between the relative LC3B and p62 expression at individual motor neurons considering age, motor neuron pool and sex (Fig. 6). For relative LC3 and p62 fluorescence measurements, Z-scores were calculated from the overall mean and SD for all animals (Table 1 provides mean ± 95% CI relative expression for each group). Examining the correlation between LC3 and p62 expression facilitates evaluation of aging effects on the various phases of autophagy (Fig. 1). Overall, there is increased autophagy in both phrenic and non-phrenic motor neurons in the cervical spinal cord of 6-month old male mice when compared to older mice. In 24-month old male and female mice, increased relative expression of both autophagy markers LC3 and p62 is consistent with impaired autophagy flux (Fig. 1B).

Fig. 6.

Fig. 6.

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Correlation between LC3B and p62 relative fluorescence intensity in individual motor neurons in the cervical spinal cord of aging male and female mice. Each point represents an individual motor neuron, symbols represent animals and colors represent age groups. Z-scores were calculated using the mean and SD for all animals. Shaded areas show bivariate normal density ellipses (90% of distribution) for each age group. Male mice at 18-and 24-months of age displayed a shift to the upper left corner (impaired autophagy) when compared to the 6-month old group in both putative phrenic (A) and non-phrenic (C) motor neurons. Female mice displayed no differences across age groups in either putative phrenic (B) or non-phrenic (D) motor neurons.

Table 1.

Relative expression of the autophagy markers LC3 and p62 in individual cervical motor neurons. Data are presented as mean ± 95% CI (arbitrary units) across age, sex and motor neuron pool, and are relative to the background-subtracted gray matter immunofluorescence intensity.

Mouse
sex		 Motor
neuron pool		 Autophagy
protein		 6-Month 18-
Month		 24-
Month
Males Phrenic LC3B 2.06 ± 0.08 1.75 ± 0.08 2.42 ± 0.08
p62 1.77 ± 0.05 2.85 ± 0.13* 3.50 ± 0.12*
Non-phrenic LC3B 2.15 ± 0.05 1.71 ± 0.07 2.29 ± 0.06
p62 1.64 ± 0.03 2.79 ± 0.12* 3.35 ± 0.10*
Females Phrenic LC3B 1.71 ± 0.09 2.16 ± 0.09 2.35 ± 0.12*
p62 3.12 ± 0.11 3.21 ± 0.13 3.00 ± 0.16
Non-phrenic LC3B 1.59 ± 0.04 2.04 ± 0.08 2.22 ± 0.07
p62 3.00 ± 0.09 3.42 ± 0.11 3.17 ± 0.14
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*,

different from the 6-month old group; post hoc Tukey-Kramer HSD, p < 0.05.

4. Discussion

The results of the present study show that there is an aging effect on autophagy markers in cervical motor neurons of mice. Overall, p62 increased by ~2 fold in the whole cervical spinal cord of 24-month old mice when compared to 18-month old mice, reflecting impairment in the degradation phase of autophagy. We found additional evidence of an effect of aging on the immunoreactivity for both LC3 and p62 in cervical motor neurons that varied with motor neuron pool and sex. Increased autophagy clearance of autophagosomes in 18- compared to 24-month old mice, reflected by reduced expression of p62, appears to be a global effect on the spinal cord not restricted to motor neurons. However, by 24-months of age, increased LC3 and p62 immunoreactivity indicates autophagosome accumulation and autophagy impairment in cervical motor neurons. These effects are somewhat different across motor neuron pools in the cervical spinal cord, with more pronounced accumulation of autophagosome markers in phrenic motor neurons. Sex differences in autophagy at cervical motor neurons across aging are consistent with estrogen effects in young mice (Xiang et al., 2019; Yang and Klionsky, 2009). Taken together, our results provide robust evidence that autophagy is impaired with age in cervical motor neurons of mice, supporting a mechanistic role for autophagy in aging-related neuromuscular dysfunction.

Aging is a series of processes characterized by alterations in multiple tissues that lead to physiological dysfunction and increased vulnerability to death. Indeed, there are several cellular and molecular hallmarks of aging including accumulation of damaged cytoplasmic structures such as proteins or organelles (Lopez-Otin et al., 2013). The importance of this accumulation and/or aggregation is noteworthy in postmitotic cells such as motor neurons (Ashrafi et al., 2014), because aggregates likely produce cellular dysfunction that is mitigated by cell division (Loos et al., 2017; Powers et al., 2009; Rubinsztein et al., 2011). Accordingly, post-mitotic cells rely on various mechanisms to cope with the accumulation of damaged cytoplasmic structures, including autophagy (Flake et al., 2015).

Autophagy is a tightly regulated, multistep process (Perluigi et al., 2015; Ravikumar et al., 2010; Yang and Klionsky, 2010) that clears damaged cytoplasmic structures. The failure of autophagy and consequent accumulation of damaged cytoplasmic structures has been linked to aging and several aging-related neurodegenerative conditions (Powers et al., 2009; Ruegsegger and Saxena, 2016), including amyotrophic lateral sclerosis (Castillo et al., 2013), Alzheimer’s disease (Lipinski et al., 2010; Pickford et al., 2008) and Parkinson’s disease (Lynch-Day et al., 2012). Importantly, dysregulated autophagy was specifically linked to neurodegeneration in these conditions (Frake et al., 2015; Milisav et al., 2015; Nah et al., 2015; Pickford et al., 2008). Autophagy impairment was also associated with aging and dysfunction of tissues other than the central nervous system including skeletal muscles (Jiao and Demontis, 2017) and neuromuscular junctions (Carnio et al., 2014; Khan et al., 2014), as well as the heart (Shirakabe et al., 2016). Available evidence supports aging-associated deficits in autophagy (Martinez-Lopez et al., 2015; Nikoletopoulou et al., 2015; Rubinsztein et al., 2011), with preclinical models showing that inhibition of autophagy results in aging phenotypes (Gonzalez Porras et al., 2018).

Impaired autophagy can result from deficits in the initiation, elongation and degradation phases that are reflected in the relative protein expression levels for these various markers (Fig. 1). For instance, models of Alzheimer’s disease show aging-related reductions in expression of the key initiation factor Becn-1 in both mice (Pickford et al., 2008) and humans (Rohn et al., 2011). Furthermore, a genome-wide analysis showed aging-related effects on autophagy of the human brain, both in Alzheimer’s disease and normal aging, with downregulation of Becn-1, as well as of the key elongation factors ATG7 and ATG5 (Lipinski et al., 2010). In a rat model of traumatic brain injury, increased expression of Becn-1 was associated with increased autophagy (Huang et al., 2018). Induction of autophagy and associated higher expression of Becn-1, LC3-II/I ratio, and decreased p62 expression, were observed in models aiming to mitigate aging effects (e.g., via caloric restriction) (Wang and Miller, 2012; Yang et al., 2014). In the present study, across a period with a significant reduction in survival (75% at 24 months of age), there was no evidence of large scale changes in the expression of Becn-1, ATG7 or ATG5/12 (Fig. 3) suggesting minimal changes in the initiation or elongation phases of autophagy in the cervical spinal cord of aging mice (Fig. 1).

With aging of the phrenic motor system, several effects initiate at the motor neuron level, with subsequent effects on diaphragm muscle atrophy and force generation. Compared to early adulthood (6-months of age; 100% survival in rodents), aging is associated with loss of ~25% of phrenic motor neurons (Fogarty et al., 2018), denervation of diaphragm muscle fibers of ~20% (Fogarty et al., 2018; Greising et al., 2015d), and impaired neuromuscular transmission by ~30%, all of which are evident early in old age (by 18-months of age; 90% survival) in both mice (Greising et al., 2015a) and rats (Fogarty et al., 2019). Although the results of this study provide evidence suggesting that in early old age there are no major effects of aging on autophagy at the cervical spinal cord level, there is evidence of autophagy being impaired in cervical motor neurons, with accumulation of p62 at 18-months of age when compared to 6-months. These findings are consistent with cell-specific differences in aging effects, and motor neurons being differentially affected compared to other cells in the spinal cord. It is possible that impairments in autophagy at cervical motor neurons contribute to their loss as well as to early deficits in neuromuscular function associated with aging.

In the present study, we found a sex difference in autophagy marker expression in the cervical spinal cord and in motor neurons. Female mice displayed higher relative p62 fluorescence intensity in early adulthood (6-months of age) than males with no difference in the relative expression across age groups for all cervical motor neurons. These results suggest that in early adulthood females have lower basal levels of autophagy than males. Indeed, previous reports indicate interactions between autophagy and sex hormone signaling pathways (Xiang et al., 2019; Yang and Klionsky, 2009), with evidence that premenopausal females display reduced autophagy due to sex hormones (Xiang et al., 2019), including in motor neurons (Olivan et al., 2014). Of note, there are no major sex differences in aging effects on the phrenic motor system or respiratory outcomes (Greising et al., 2015b), including phrenic motor neuron loss (Fogarty et al., 2018), reductions in diaphragm muscle specific force or myofiber cross-sectional area (Khurram et al., 2018) or reductions in maximal transdiaphragmatic pressure (Pareja-Cajiao et al., 2020). Taken together, these studies support impairments in autophagy as a common mechanism of aging-related neuromuscular dysfunction.

Neuromuscular transmission failure occurs well before a loss of force is noticeable (Greising et al., 2015b, 2017). In fact, aging effects on diaphragm neuromuscular transmission in mice that are present in early old age (18-months of age; 90% survival) persist into old age (24-months of age; 75% survival) (Greising et al., 2015a). Of note, diaphragm sarcopenia (muscle fiber atrophy and reduced maximum specific force) only becomes evident by 24-months of age in mice (Greising et al., 2013, 2015b, 2015c, 2017) and rats (Elliott et al., 2016b). Additionally, phrenic motor neuron loss is also evident in 24-month old rats (Fogarty et al., 2018). These findings suggest that functional loss and sarcopenia are preceded by a period of susceptibility to aging effects, likely at the motor neuron level. The expression levels of p62 increased by ~2 times in the 24-month old group when compared to the 18 month-old group, suggesting an overall decrease in autophagy at the cervical spinal cord. At the motor neuron level, there is an aging-related impairment in autophagy compared to young animals as indicated by the increase in LC3 levels compared to both 6- and 18-months of age, and by the increase in p62 compared to the 6-month old group. Accordingly, the combined accumulation of both LC3 and p62 by 24-months of age represents a further impairment in autophagy compared to the 18-month old animals. The functional consequences of impaired autophagy on motor neuron function are certainly intriguing and deserve further exploration. Although the physiological aging of the phrenic motor system is well characterized (Elliott et al., 2016a; Fogarty et al., 2018, 2019; Greising et al., 2013, 2015d, 2017), aging effects on non-phrenic motor neurons (e.g., limb muscle motor pools) in the cervical spinal cord are not well understood. Indeed, our findings are consistent with evidence of differential aging effects across motor neuron pools (Hashizume and Kanda, 1995).

In summary, the aging-related changes in autophagy observed in the present study likely contribute to the neuromuscular dysfunction associated with aging of the phrenic motor system. Taken together, the aging-related changes in expression of LC3B and p62 at phrenic motor neurons suggest that autophagy is reduced at 18-months of age, with progressive autophagosome accumulation by 24-months of age. The differential expression of autophagy markers in putative phrenic motor neurons vs. non-phrenic motor neurons also highlights the importance of evaluating autophagy across motor neuron pools. In the present study, there was an effect of motor neuron pool on the expression of both LC3 and p62, with phrenic motor neurons displaying higher relative fluorescence intensity than non-phrenic motor neurons across age groups. Whether differences in accumulation of LC3 and p62 in phrenic motor neurons underlie their susceptibility to aging effects should be explored directly.

Footnotes

Declaration of competing interest

This work was funded by NIH grants R01 AG044615 and R01 AG057052, and the Mayo Clinic.

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