Abstract
Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1G93A mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1G93A rats, numerous cells displayed nuclear p16INK4a as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16INK4a-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16INK4a-positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1G93A rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days in vitro and subsequent passages. Senescent SOD1G93A microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-β-Gal) activity as well as positive labeling for p16INK4a, p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16INK4a staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1G93A rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by progressive upper and lower motor neuron degeneration, leading to muscle weakness and paralysis (Tsai et al, 2017)
A fundamental feature of cellular senescence is the arrest of the cell cycle through p16INK4A-mediated pathway, which is usually associated with p53 nuclear expression (Prieur et al, 2011). p53 becomes activated in response to a variety of cellular stressors including DNA damage and oxidative stress leading to an increased half-life of the p53 protein, phosphorylation and nuclear translocation
Based on a previous report showing an increase of p16INK4a RNA levels in symptomatic SOD1G93A rats (Das and Svendsen, 2015), we examined the number of p16INK4a-positive nuclei and Lamin B1 expression in the ventral horn of the lumbar cord during paralysis progression
Summary
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by progressive upper and lower motor neuron degeneration, leading to muscle weakness and paralysis (Tsai et al, 2017). Glial cells become activated, proliferate and display inflammatory features characteristic of gliosis (Philips and Rothstein, 2014; Trias et al, 2018a) These kinds of cellular stresses combined with DNA damage or strong mitogenic signaling in vulnerable cells have the potential to induce cellular senescence (Rodier et al, 2009), a basic and heterogenous mechanism by which damaged cells adapt to maintain survival and prevent potentially deleterious expansion or oncogenic transformation during aging (Munoz-Espin and Serrano, 2014). Nuclear expression of p16INK4A is considered a robust molecular marker of cellular aging, as its expression increases in a variety of aged tissues (Baker et al, 2011) Another remarkable senescence-associated marker is the loss of nuclear Lamin B1 (Freund et al, 2012), which together with other lamins, is essential to maintain nucleus stability, size and shape (Dechat et al, 2008). The loss of nuclear Lamin B1 in particular is recognized as a senescence marker, functionally associated with the induction of p16INK4A and p53 (Freund et al, 2012)
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