Richard Robitaille

Background: Aging is associated with progressive neuromuscular junction (NMJ) degeneration. The C-terminal agrin fragment (CAF), a circulating product of agrin cleavage, has been proposed as a minimally invasive biomarker of NMJ degradation, but its relationships with specific neuromuscular and functional characteristics, and the potential modulating role of physical activity, remain poorly defined. Objective: To examine age- and physical activity-related differences in serum CAF and to test its associations with neuromuscular integrity and functional capacity across the adult male lifespan. Methods: We studied 136 community-dwelling men aged 20-92 years (active, n=87; inactive, n=49). Serum CAF was measured by ELISA; NCAM-positive fiber proportion, a widely used marker of myofiber denervation, was assessed by immunolabeling of vastus lateralis biopsies (n=127). Neuromuscular assessments included maximal voluntary isometric contraction (MVIC), lower-limb muscle power, electromechanical delay (EMD), nerve conduction velocities, H/M ratio, central activation ratio, co-activation, and twitch characteristics. Functional capacity was assessed with the 6-minute walk test, alternating step test, Timed Up and Go, sit-to-stand tests, and fast gait speed. Results: Serum CAF increased progressively with age (β= 21,51, p<0.001) and was positively associated with NCAM-positive fiber proportion (β= 119,5 p=0.01). Higher CAF was associated with lower MVIC (β=-0.01, p=0.026), reduced lower-limb power (β= -0.01, p=0.027), and poorer performance on the 6MWT, alternating step test, TUG, sit-to-stand, and gait speed (p<0.05). CAF was associated with longer EMD in the whole cohort (β= 0.003, p=0.017) and inactive men (β= 0.005, p=0.007), but not in active men, and with lower H/M ratio in inactive men only (β= -0.003, p=0.0217). No associations were found with nerve conduction velocities, central activation ratio, co-activation, or twitch amplitude. Active men showed lower CAF than inactive men, particularly after age 70 (p=0.023). Conclusion: In community-dwelling men, serum CAF is associated with age-related declines in neuromuscular and functional capacity and shows tissue-level consistency with the proportion of NCAM-positive fibers, supporting its use as an accessible biomarker of NMJ remodeling. Its limited association with most electrophysiological measures indicates it captures a specific component of neuromuscular aging rather than a global index of neuromuscular function. Physical activity was associated with a more favorable CAF profile, particularly among older men, identifying physical activity as a potential modifiable factor in age-related NMJ decline. Highlights: Serum CAF rises progressively across the adult lifespan in men. CAF levels correlate with muscle fiber denervation markers. Elevated CAF is linked to poorer neuromuscular and physical function. Physical activity is associated with a more favorable CAF profile.

Nicotinamide adenine dinucleotide (NAD⁺) depletion is proposed to drive muscle aging, yet its human relevance remains unresolved. NAD(H), NADP(H) and glutathione were quantified in biopsies from 137 men aged 20-93y. NAD(P) pools were not associated with age, whereas glutathione expanded without altered GSH/GSSG. NAD⁺ and NADPH tracked performance, muscle mass and mitochondrial content beyond age, indicating that NAD(P) metabolism reflects retained muscle health more than aging and arguing against obligatory age-related NAD(P) depletion.

The neuromuscular junction (NMJ) is a specialized synapse essential for effective motor neuron–muscle communication and is increasingly recognized as a vulnerable site in aging and neuromuscular disease. While traditionally considered a final common pathway for motor deficits, accumulating evidence demonstrates that NMJ dysfunction is an early and critical driver of disease onset and progression in conditions such as amyotrophic lateral sclerosis and Charcot–Marie–Tooth disease. This review highlights shared and disease-specific mechanisms contributing to NMJ impairment, including presynaptic, postsynaptic, and perisynaptic Schwann cell defects in these diseases. We also discuss age-related changes at the NMJ, emphasizing its role in sarcopenia and muscle weakness in older adults. Furthermore, we explore emerging molecular drivers of NMJ dysfunction uncovered through studies in congenital myasthenic syndromes, autoimmune disorders, and advanced omics approaches. By integrating insights across diseases and aging, we underscore the potential for shared therapeutic strategies aimed at stabilizing NMJ function. Promising interventions targeting presynaptic neurotransmitter release, postsynaptic excitability, and perisynaptic Schwann cells are discussed as avenues to improve neuromuscular transmission and maintain muscle strength. Finally, we discuss the challenges and opportunities in translating these mechanistic insights into clinical therapies and highlight how novel human neuromuscular organoid models and advanced molecular profiling can bridge this gap. Together, these insights establish the NMJ as a critical, modifiable target for preserving motor function across neuromuscular diseases and aging.

Astrocytes support neuron function through a range of regulatory mechanisms, including synaptic modulation. There is a more comprehensive understanding of astrocyte contribution to transmission at excitatory synapses than inhibitory synapses. However, the synaptic activity of inhibitory neurons has extensive consequences on neuron activity, circuitry, brain states and function, which is consolidated by the inherent diversity of GABAergic inhibitory neurons. This review provides an overview of the purposeful function of astrocytes at the synapses of GABAergic inhibitory neurons at structural, ionic, molecular, circuit, and behavioral levels and incorporates diversity into the current understanding of inhibitory tripartite synapses.

Astrocyte functional heterogeneity within a given neuronal circuit remains largely undetermined, particularly their role at tripartite synapses. Here, we examine multiple functional characteristics of astrocytes distinguished by their specific spatial relation to inhibitory synapses made on distinct hippocampal CA1 pyramidal cell domains: astrocytes covering the peri-somatic area in stratum pyramidale (SP) receiving input from Parvalbumin interneurons, or the apical dendritic area in stratum radiatum (SR) innervated by inhibitory inputs from Somatostatin interneurons. Whole-cell dye-filling and confocal imaging of SR astrocytes showed a typical bushy organization of processes while those of SP astrocytes were more polarized, indicating astrocyte morphological heterogeneity. In addition, SP astrocytes formed a smaller, yet polarised syncytium and displayed greater input resistance relative to SR astrocytes. The two populations of astrocyte are functionally different as indicated by their intrinsic Ca ²⁺ signaling properties: SP astrocyte Ca ²⁺ events had a lower frequency and temporal density, but greater amplitude, relative to SR astrocytes. Using the territorial segregation of inhibitory synapses, we observed that the selective activation using DREADD or blockade with intracellular BAPTA of the two populations of astrocytes regulated inhibitory synapses exclusively in their own syncytial territory. Furthermore, each astrocyte population selectively mediated long-term depression at the respective inhibitory synapses through Ca ²⁺ -dependent modulation of post-synaptic targets. These results indicate domain-specific regulation of inhibitory synapses by distinct SP and SR astrocyte syncytia. Transcriptional analysis revealed enriched gene expression in SP relative to SR astrocytes, notably for regulation of cell growth and morphology, synaptic function and signaling. Overall, our findings reveal a functional specialization of astrocyte subtypes in the hippocampus, highlighting heterogeneous astrocyte regulation of hippocampal synaptic networks important for learning and memory. Graphical

In response to various stressors, cells can enter a state called cellular senescence which is characterized by irreversible cell cycle arrest and a senescence‐associated secretory phenotype (SASP). The progressive accumulation of senescent glial cells in the central nervous system (CNS) with aging suggests a potential role for senescence as driver of aging and inflammation in the brain. As the main immune cell population residing in the CNS, microglia are thought to play a pivotal role in the progression of age‐associated neuroinflammation. Furthermore, due to their slow turnover, microglia are highly susceptible to undergoing cellular senescence. However, current understanding of age‐related changes in microglia and their impact on brain aging is limited. Due to the challenge in accessing human primary microglia and the lack of models to adequately recapitulate aging, this knowledge is predominantly limited to rodent studies. Here, we chemically induced senescence in a human immortalized microglia cell line with a cocktail of senescence‐inducing molecules. We demonstrate that chemically induced senescent microglia adopt a proinflammatory phenotype, have reduced phagocytic activity, and impaired calcium activity. Our results show that chemically induced senescence can mimic features of cellular aging and can provide insight into the impact of aging and cellular senescence on human microglia.

The neuromuscular junction (NMJ) is a highly reliable synapse to carry the control of the motor commands of the nervous system over the muscles. Its development, organization, and synaptic properties are highly structured and regulated to support such reliability and efficacy. Yet, the NMJ is also highly plastic, able to react to injury, and able to adapt to changes. This balance between structural stability and synaptic efficacy on one hand and structural plasticity and repair on another hand is made possible by perisynaptic Schwann cells (PSCs), glial cells at this synapse. They regulate synaptic efficacy and structural plasticity of the NMJ in a dynamic, bidirectional manner owing to their ability to decode synaptic transmission and by their interactions with trophic-related factors. Alteration of these fundamental roles of PSCs is also important in the maladapted response of NMJs in various diseases and in aging.

In response to various stressors, cells can enter a state called cellular senescence which is characterized by irreversible cell cycle arrest and a senescence-associated secretory phenotype (SASP). The progressive accumulation of senescent glial cells in the central nervous system (CNS) with aging suggests a potential role for senescence as driver of aging and inflammation in the brain. As the main immune cell population residing in the CNS, microglia are thought to play a pivotal role in the progression of age-associated neuroinflammation. Furthermore, due to their slow turnover, microglia are highly susceptible to undergoing cellular senescence. However, current understanding of age-related changes in microglia and their impact on brain aging is limited. Due to the challenge in accessing human primary microglia and the lack of models to adequately recapitulate aging, this knowledge is predominantly limited to rodent studies. Here, we chemically induced senescence in a human immortalized microglia cell line with a cocktail of senescence-inducing molecules. We demonstrate that chemically induced senescent microglia adopt a pro-inflammatory phenotype, have reduced phagocytic activity and impaired calcium activity. Our results show that chemically induced senescence can mimic features of cellular aging and can provide insight on the impact of aging and cellular senescence on human microglia.