Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age‐related morphological changes

Abstract

SummaryNeuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age‐related changes. Aging effects on the NMJ components—motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)—have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild‐type mice, all NMJ components showed age‐associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain‐specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic‐specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age‐related changes via sympathetic innervation.

What is Normal? Neuromuscular junction reinnervation after nerve injury

“…The presence of increasing motor endplate fragmentation was unexpected, particularly in the setting of progressive NMJ reinnervation. Previous studies from our lab and others have shown endplate fragmentation occurs with aging . In the present study, age did not contribute to endplate fragment number in uninjured mice; the average number of fragments in uninjured mice was 3 or 4 at all time‐points, and all mice were under 10 months of age.…”

“…After fixation of whole muscles, the four component tendons of the EDL were dissected out in cold PBS solution. Immunofluorescent staining was performed as described elsewhere . All muscles were washed in PBS and incubated in blocking buffer (5% normal goat serum, 2% Triton X‐100, 5% bovine serum albumin in PBS) for 1 hour at room temperature.…”

“…Immunofluorescent staining was performed as described elsewhere. 10 All muscles were washed in PBS and incubated in blocking buffer (5% normal goat serum, 2% Triton X-100, 5% bovine serum albumin in PBS) for 1 hour at room temperature. Tissues were then stained with primary antibodies, rabbit anti-neurofilament 200 (NF200; 1:500; Millipore Sigma, St Louis, Missouri) or rabbit anti-S100b (1:1000; DAKO North America, Via Real, Carpinteria, California), overnight at 4 C. After rinsing in washing buffer (PBS with 0.2% Triton X-100) for 45 minutes, muscles were incubated with goat anti-rabbit immunoglubulin G-Alexa Fluor 488 (1:1000; Invitrogen-Molecular Probes, Carlsbad, California) for 1 hour at room temperature.…”

Gpr126/Adgrg6 contributes to the terminal Schwann cell response at the neuromuscular junction following peripheral nerve injury

“…At 1, 3, or 6 weeks following nerve transection and repair, SM from both the injured and sham uninjured sides were harvested under the dissecting microscope. For immunostaining, SM muscles ( n = 5–6 mice/genotype) were weighed, immediately placed in cold 0.1 M phosphate buffered saline (PBS, pH 7.4), and then fixed in 4% paraformaldehyde as described before (Snyder‐Warwick, Satoh, Santosa, Imai, & Jablonka‐Shariff, ). Muscles were cryoprotected in 15 and 30% sucrose/PBS, frozen, and sectioned.…”

“…NMJs were immunofluorescently stained as previously described in detail (Snyder‐Warwick et al, ). Briefly, frozen sections (25 μm thick) or whole mounts of the SM or EDL muscles were washed with PBS, incubated for 60 minutes in blocking buffer (containing 5% normal goat serum, 2% Triton‐X 100, 5% BSA) and then incubated in primary antibodies overnight at 4°C.…”