Role of Agrin in tissue repair and regeneration: From mechanisms to therapeutic opportunities (Review).

Tissue regeneration is a complex process that involves the recruitment of various types of cells for healing after injury; it is mediated by numerous precise interactions. However, the identification of effective targets for improving tissue regeneration remains a challenge. As an extracellular matrix protein, Agrin plays a critical role in neuromuscular junction formation. Furthermore, recent studies have revealed the role of Agrin in regulating tissue proliferation and regeneration, which contributes to the repair process of injured tissues. An in­depth understanding of the role of Agrin will therefore be of value. Given that repair and regeneration processes occur in various parts of the human body, the present systematic review focuses on the role of Agrin in typical tissue and highlights the potential signaling pathways that are involved in Agrin­induced repair and regeneration. This review offers important insight into novel strategies for the future clinical applications of Agrin­based therapies, which may represent a feasible treatment option for patients who require organ replacement or repair.

Local protein synthesis at neuromuscular synapses is required for motor functions.

Motor neurons are highly polarized, and their axons extend over great distances to form connections with myofibers via neuromuscular junctions (NMJs). Local translation at the NMJs in vivo has not been identified. Here, we utilized motor neuron-labeled RiboTag mice and the TRAP (translating ribosome affinity purification) technique to spatiotemporally profile the translatome at NMJs. We found that mRNAs associated with glucose catabolism, synaptic connection, and protein homeostasis are enriched at presynapses. Local translation at the synapse shifts from the assembly of cytoskeletal components during early developmental stages to energy production in adulthood. The mRNA of neuronal Agrin (Agrn), the key molecule for NMJ assembly, is present at motor axon terminals and locally translated. Disrupting the axonal location of Agrn mRNA causes impairment of synaptic transmission and motor functions in adult mice. Our findings indicate that spatiotemporal regulation of mRNA local translation at NMJs plays critical roles in synaptic transmission and motor functions in vivo.

Metformin improves skeletal muscle and physical capacity by stabilizing neuromuscular junction in older adults.

OBJECTIVES: Metformin is an anti-diabetic drug with protective effects on skeletal muscle and physical capacity. However, the relevant mechanisms of action on skeletal muscle remain poorly understood. We investigated the potential contribution of neuromuscular junction (NMJ) degradation to skeletal muscle and physical capacity in geriatric men taking metformin. METHOD: We recruited geriatric men for placebo (Age=73.1 ± 4.2 years, n = 70) and metformin (Age=70.1 ± 4.5 years, n = 62) groups. The patients in the metformin group received 1700 mg of metformin twice a day for 16 weeks. We measured plasma c-terminal agrin-fragment-22 (CAF22) and neurofilament light chain (NfL) as markers of neuromuscular junction (NMJ) degradation and neurodegeneration, respectively, with relevance to handgrip strength (HGS) and short physical performance battery (SPPB; a marker of physical capacity) in older adults taking metformin. These findings were associated with reduced oxidative stress in the metformin group. RESULTS: At baseline, both groups had similar HGS, gait speed, SPPB scores, and plasma biochemistry. Metformin improved HGS, gait speed, and cumulative SPPB scores in geriatric men (all p < 0.05). Metformin also reduced plasma CAF22 and NfL levels when compared to baseline. Similar observations were not found in the placebo group. Correlation analysis revealed significant correlations of plasma CAF22 with HGS, gait speed, and cumulative SPPB scores in the metformin group. These observations were associated with reduced oxidative stress in the metformin group. CONCLUSION: Altogether, the restorative effects of metformin on skeletal muscle and physical capacity involve NMJ stabilization. Our data is clinically relevant for geriatric men with functional disabilities.

Tracking the Plasma C-Terminal Agrin Fragment as a Biomarker of Neuromuscular Decline in 18- to 87-Year-Old Men.

OBJECTIVES: Plasma C-terminal agrin-fragment-22 (CAF22), a breakdown product of neuromuscular junction, is a potential biomarker of muscle loss. However, its levels from adolescence to octogenarians are unknown. METHODS: We evaluated young (18-34 years, n = 203), middle-aged (35-59 years, n = 163), and old men (60-87 years, n = 143) for CAF22, handgrip strength (HGS), appendicular skeletal-mass index (ASMI), and gait speed. RESULTS: We found an age-associated increase in CAF22 from young (100.9 ± 29 pmol) to middle-aged (128.3 ± 38.7 pmol) and older men (171.5 ± 35.5 pmol) (all p<0.05). This was accompanied by a gradual reduction in HGS (37.7 ± 6.1 kg, 30.2 ± 5.2 kg, and 26.6 ± 4.7 kg, for young, middle-aged, and old men, respectively), ASMI (8.02 ± 1.02 kg/m2, 7.65 ± 0.92 kg/m2, 6.87 ± 0.93 kg/m2, for young, middle-aged, and old men, respectively), and gait speed (1.29 ± 0.24 m/s, 1.05 ± 0.16 m/s, and 0.81 ± 0.13 m/s, for young, middle-aged, and old men, respectively). After adjustment for age, we found negative regressions of CAF22 with HGS (- 0.0574, p < 0.001) and gait speed (- 0.0162, p < 0.001) in the cumulative cohort. The receiver operating characteristics analysis revealed significant efficacy of plasma CAF22 in diagnosing muscle weakness (HGS < 27 kg) (middle-aged men; AUC = 0.731, 95% CI = 0.629-0.831, p < 0.001, Older men; AUC = 0.816, 95% CI = 0.761-0.833, p < 0.001), and low gait speed (0.8 m/s) (middle-aged men; AUC = 0.737, 95% CI = 0.602-0.871, p < 0.001, older men; AUC = 0.829, 95% CI = 0.772-0.886, p < 0.001), and a modest efficacy in diagnosing sarcopenia (middle-aged men; AUC = 0.701, 95% CI = 0.536-0.865, p = 0.032, older men; AUC = 0.822, 95% CI = 0.759-0.884, p < 0.001) in middle-aged and older men. CONCLUSION: Altogether, CAF22 increases with advancing age and may be a reliable marker of muscle weakness and low gait speed.

Prostaglandin E2 Induces YAP1 and Agrin Through EP4 in Neonatally-Derived Islet-1+ Stem Cells.

Prostaglandin E2 (PGE2) has recently gained attention in the field of regenerative medicine because of the beneficial effects of this molecule on stem cell proliferation and migration. Furthermore, PGE2 has the ability to mitigate immune rejection and fibrosis. In the colon and kidney, PGE2 induces YAP1, a transcription factor critical for cardiac regeneration. Establishing a similar connection in stem cells that can be transplanted in the heart could lead to the development of more effective therapeutics. In this report, we identify the effects of PGE2 on neonatal Islet-1+ stem cells. These stem cells synthesize PGE2, which functions by stimulating the transcription of the extracellular matrix protein Agrin. Agrin upregulates YAP1. Consequently, both YAP1 and Agrin are induced by PGE2 treatment. Our study shows that PGE2 upregulated the expression of both YAP1 and Agrin in Islet-1+ stem cells through the EP4 receptor and stimulated proliferation using the same mechanisms. PGE2 administration further elevated the expression of stemness markers and the matrix metalloproteinase MMP9, a key regulator of remodeling in the extracellular matrix post-injury. The expression of PGE2 in neonatal Islet-1+ cells is a factor which contributes to improving the functional efficacy of these cells for cardiac repair.

Plasma C-terminal agrin fragment concentrations across adulthood: Reference values and associations with skeletal muscle health.

BACKGROUND: Increasing interest surrounds the utility of blood-based biomarkers for diagnosing sarcopenia. C-terminal agrin fragment (CAF), a marker of neuromuscular junction stability, is amongst the most promising candidates; however, a dearth of reference data impedes the incorporation of its use in public health settings. This study aimed to establish reference values for plasma CAF concentrations across adulthood in a large, well-characterized cohort of healthy adults; and comprehensively examine the association between plasma CAF levels and skeletal muscle health. METHODS: One thousand people aged between 18 and 87 years took part in this study (mean age = 50.4 years; 51% females). Body composition and muscle strength were examined using DXA and hand dynamometry. Plasma CAF concentrations were measured, in duplicate, using commercially available ELISA kits. Sarcopenia and individual sarcopenia signatures [low skeletal muscle index (SMI) only/low grip strength only] were classified using the EWGSOP2 algorithm. RESULTS: Detailed reference CAF values, according to sex and age, are presented. A significant but modest age-related increase in plasma CAF concentration was observed (P = 0.018). Across adulthood, CAF concentrations were negatively associated with grip strength and SMI (both P < 0.001). In people ≥50 years old, CAF concentrations were 22.6% higher in those with sarcopenia (P < 0.001), 11.3% higher in those with low SMI (P = 0.006) and 9.6% higher in those with low grip strength (P = 0.0034), compared with controls. People in the highest CAF concentration quartile, had 3.25 greater odds for sarcopenia (95% CI = 1.41-7.49, P = 0.005), 2.76 greater odds for low SMI (95% CI = 1.24-5.22, P = 0.012), and 2.56 greater odds for low grip strength (95% CI = 1.07-5.57, P = 0.037), compared with those in the lowest quartile. People with a CAF Z-score ≥2 had 9.52 greater odds for sarcopenia (95% CI = 3.01-30.05, P < 0.001) compared with a Z-score <1. Plasma CAF concentration had an acceptable level of diagnostic accuracy for sarcopenia (AUC = 0.772, 95% CI = 0.733-0.807, P < 0.001). CONCLUSIONS: The reference values presented herein may guide the clinical interpretation of circulating CAF and help identify people at risk of poor skeletal muscle outcomes for inclusion in therapeutic interventions. Our findings add clarity to existing data, demonstrating a robust relationship between circulating CAF and skeletal muscle integrity in the largest adult cohort to date, and support the use of CAF as an accessible, cost-effective screening tool for sarcopenia. However, further research into the prognostic utility of plasma CAF, and the establishment of normative data from other populations, are urgently needed if routine CAF screening is to be embedded into public healthcare settings.

Building, Breaking, and Repairing Neuromuscular Synapses.

A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.

The MuSK agonist antibody protects the neuromuscular junction and extends the lifespan in C9orf72-ALS mice.

The disassembly of the neuromuscular junction (NMJ) is an early event in amyotrophic lateral sclerosis (ALS), ultimately leading to motor dysfunction and lethal respiratory paralysis. The hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most common genetic mutation, and the dipeptide repeat (DPR) proteins have been shown to cause neurodegeneration. While no drugs can treat ALS patients efficiently, new treatment strategies are urgently needed. Here, we report that a MuSK agonist antibody alleviates poly-PR-induced NMJ deficits in C9orf72-ALS mice. The HB9-PRF/F mice, which express poly-PR proteins in motor neurons, exhibited impaired motor behavior and NMJ deficits. Mechanistically, poly-PR proteins interacted with Agrin to disrupt the interaction between Agrin and Lrp4, leading to attenuated activation of MuSK. Treatment with a MuSK agonist antibody rescued NMJ deficits, and extended the lifespan of C9orf72-ALS mice. Moreover, impaired NMJ transmission was observed in C9orf72-ALS patients. These findings identify the mechanism by which poly-PR proteins attenuate MuSK activation and NMJ transmission, highlighting the potential of promoting MuSK activation with an agonist antibody as a therapeutic strategy to protect NMJ function and prolong the lifespan of ALS patients.

Lipid-Lowering Medications are Associated with Reduced Sarcopenia-Related Quality of Life in Older Adults with Hyperlipidemia.

PURPOSE: Statins medications negatively affect age-associated loss of muscle mass and strength, termed sarcopenia, and neuromuscular junction (NMJ) integrity. However, their association with the sarcopenia-related-quality-of-life (SarQoL) is unknown. METHODS: In this cross-sectional, case control study, we recruited male nonusers (n = 75 and age 75.2 ± 5.9 years) and users (n = 77 and age 77.1 ± 6.2 years) of statins to evaluate SarQoL and handgrip strength (HGS). We also measured plasma C-terminal agrin fragment-22 (CAF22) as a marker of NMJ degradation. RESULTS: Statin users had higher CAF22, and lower HGS, and cumulative SarQoL scores than non-users (all p < 0.05). Plasma CAF22 exhibited negative correlations with SarQoL scores for physical and mental health, locomotion, functionality, activities-of-daily-living, and cumulative SarQoL in statins users and non-users (all p < 0.05). Lastly, the cumulative SarQoL scores exhibited positive associations with HGS and gait speed in the study participants (all p < 0.05). CONCLUSIONS: Collectively, statin usage was associated with NMJ degradation and reduced SarQoL. Statins should be cautiously prescribed in patients with sarcopenia with reduced QoL.

Deficiency of skeletal muscle Agrin contributes to the pathogenesis of age-related sarcopenia in mice.

Sarcopenia, a progressive and prevalent neuromuscular disorder, is characterized by age-related muscle wasting and weakening. Despite its widespread occurrence, the molecular underpinnings of this disease remain poorly understood. Herein, we report that levels of Agrin, an extracellular matrix (ECM) protein critical for neuromuscular formation, were decreased with age in the skeletal muscles of mice. The conditional loss of Agrin in myogenic progenitors and satellite cells (SCs) (Pax7 Cre:: Agrin flox/flox) causes premature muscle aging, manifesting a distinct sarcopenic phenotype in mice. Conversely, the elevation of a miniaturized form of Agrin in skeletal muscle through adenovirus-mediated gene transfer induces enhanced muscle capacity in aged mice. Mechanistic investigations suggest that Agrin-mediated improvement in muscle function occurs through the stimulation of Yap signaling and the concurrent upregulation of dystroglycan expression. Collectively, our findings underscore the pivotal role of Agrin in the aging process of skeletal muscles and propose Agrin as a potential therapeutic target for addressing sarcopenia.