To clarify whether senescence-associated phenomena and TDP-43 dysfunction could be implicated in ALS, we measured the abovementioned variables in the familial ALS transgenic mouse model hSOD1-G93A at different disease stages. The senescence markers p16 and p21, typical biomarkers of senescent cells , were analyzed in LSC. Using two different technics (IHC and IF), we characterized the cellular expression pattern of p16. The results show that the expression of p16 mRNA was progressively increased during disease evolution (Fig. 1a), whereas p21 mRNA levels were only higher at the end-stage (Fig. 1b). p16 and p21 exhibited a predominantly cytoplasmic pattern (Fig. 1c). As shown by IF, p16 positive cells were microglia (Iba1+ cells) (Fig. S1a) and astroglia (GFAP+ cells) (Fig. S1b). These results indicate dynamic changes in cellular senescence-associated markers and SASP related to disease evolution. p16 expression is highly expressed before the symptomatology in our transgenic mice. This fact suggests a role for p16 in disease initiation and progression. Interestingly, senescence-associated cell cycle arrest in an early symptomatic stage (120d) is driven exclusively by p16, whereas p21 only increases later in this model. This may be seen as a result of the late-onset activation of p53 and the DNA damage response pathway, similar to what occurs in the senescence process in microglia. In contrast to p21 (related to reversible cell cycle arrest or quiescence), the senescence process depends heavily on prolonged p16 expression. Strictly speaking, our work and most published articles on ‘senescence’ do not demonstrate an always irreversible cell cycle arrest. There may be divergent processes sharing common biomarkers. This is the case with macrophage polarization, in which p16 expression and SA-β-gal activity are physiological, reversible, and not associated with cellular senescence . In this line, cytoplasmic p16 can regulate cell migration in a manner similar to cyclin D1. This evidence reflects a convergent pathway of cell cycle- and senescence-associated proteins regulating cytoskeleton functions. In the case of ALS, cytoskeleton regulators like Rac1 and Cdc42 are implicated in the disease progression and neuroinflammation . Thus, we hypothesized that cytoplasmic p16 could have a similar role in ALS. Like p16 cytoplasmic functions, p21 inhibits the ROCK/LIMK/Cofilin Pathway through MAPK signaling, inducing cytoskeleton remodeling.
We also analyzed another senescence canonical biomarker: SA-β-gal activity. The main cellular populations expressing SA-β-gal in ventral LSC are the motor neuron cells (Nissl+ cells in the ventral horn, with a motor-neuron compatible cellular size). Neurons of other LSC locations and the vast majority of Nissl- do not show SA-β-gal activity (Fig. 1d and Fig S2). Interestingly, SA-β-gal activity was reduced during disease progression in motor neurons and in a small fraction of Nissl- cells (compatible with glia). Our findings agree with previously shown data demonstrating that SA-β-gal activity in neurons is not associated with senescence, although it is increased in aging mouse brain . Our results suggest that motor neurons contain more lysosomes in cell body than other cells, and that their biogenesis is compromised in this ALS mouse model. In this line, lysosomal mass deficit has already described in this model, highlighting a role of hSOD1 aggregates disturbing lysosomal biogenesis  and potentially explaining our results from the SA-β-gal activity assay.
Another marker commonly employed in senescence description is the increase in cytokines linked to SASP. In this case, we quantified the expression of typical SASP markers Il1a and Il6. We analyzed as well the expression of Ifna and Ifnb (corresponding to type-I IFN response) as they are postulated as late-senescence markers and could be helpful in determining senescence progression in the LSC of this model. The expression of Ifna was not detected in any of the analyzed samples (data not shown). We observed a different pattern of expression between Il1a (Fig. 1e) and Il6 (Fig. 1f). Il1a is increased in the pre-symptomatic stage and is known to be the upstream regulator of IL-6 in SASP . IL-6 is increased in cerebrospinal fluid in ALS, Alzheimer’s, and Parkinson’s disease . In contrast, Ifnb expression (Fig. 1g) is not altered, which could indicate that senescence in this model does not evolve a late phase. Overall, this might reflect a complex interaction between senescence, SASP, and changes in reactive glial cells and neurodegeneration.
Regarding TDP-43 splicing function, in mice it controls the inclusion in Adipor2 mRNA (Fig S3a,b). In line with loss of TDP-43 function in this model, cryptic exon inclusion in Adipor2 mRNA was higher in lumbar spinal cord in end-stage mice (Fig. 1h) and positively correlated with p16 expression (Fig. 1i). The present data are the first to show specific alteration regarding splicing function in this ALS model. Notably, this process is associated with an increase in the senescence marker p16, and the two processes are likely to be linked in the same pathway. Of note, increased p16 seem restricted to central nervous system, as sciatic nerve does not show these changes (Fig. S3c), in contrast with Adipor2 cryptic exon inclusion, which was also increased in sciatic nerve (Fig. S3d). Noteworthy, increased Adipor2 cryptic exon was associated with loss of Adipor2 mRNA levels, suggesting increased non-sense mediated decay in both locations (Fig. S3e).
We wanted to explore the potential benefits of senolytic treatment due to the higher expression of senescence related genes in this mouse model. We performed Navitoclax treatment following the protocol described for Alzheimer’s disease mouse model . The treatment was initiated at 90 days old and finished at end point (Figure S4a). We estimated the disease progression by weight loss. Navitoclax treatment did not prevent weight loss, neither prolonged survival (Figure S4b and 1j). Finally, we quantified senescence and SASP genes in lumbar spinal cord. None of the analyzed genes showed statistically significant differences (Figure S4c). These results suggest differences in molecular effectors between Alzheimer’s and ALS.
Navitoclax is an inhibitor of antiapoptotic protein Bcl2. Senescent cells are highly dependent of different antiapoptotic members. Senolysis is achieved when this antiapoptotic protein is inhibited, promoting cell death. Navitoclax treatment is not enough to slow the disease progression and does not extend the survival. In contrast with data in Alzheimer’s and Parkinson’s disease models, this treatment does not prevent the increase of senescence and SASP markers. It suggests that senescence phenotype is not driven by Bcl2 expression of stressed or aged cells in this model. Further studies are warranted to determine whether senescence-linked phenomena are mechanistically involved in this fatal disease, clearing the pathway for therapeutic development.