Insights into the protective role of Estrogens in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is the most common motoneuron disorder, characterized by the progressive degeneration of both upper and lower motoneurons, ultimately leading to death, mainly due to respiratory failure within an average of 36 months from onset. Epidemiological data consistently reveal a male predominance in disease incidence, a disparity that decreases with advancing age, particularly after menopause, suggesting a potential neuroprotective role for endogenous estrogens. Given that approximately 97% of ALS patients exhibit cytoplasmic aggregates of TAR DNA-binding protein 43 (TDP-43) with pathological post-translational modifications as a neuropathological hallmark, this study aims to elucidate the molecular mechanisms through which estrogens may counteract the trait of TDP-43 proteinopathy in ALS. Using a multi-model approach, this research investigates the effects of estrogens in murine motoneuron-like cells (NSC-34) expressing pathological forms of TDP-43, in lymphoblastoid cells derived from sporadic ALS (sALS) patients, and in cell-free assays employing purified recombinant human TDP-43. The results demonstrate that treatment with 17β-estradiol (E2) effectively rescues the cytoplasmic mislocalization of full-length TDP-43 in motoneuron-like cells, leading to a significant reduction of associated cellular toxicity and mitochondrial superoxide production. Moreover, in lymphoblasts derived from sALS patients, which endogenously display pathological TDP-43 mislocalization, E2 treatment restored the physiological nuclear localization of TDP-43 and reduced its pathological hyperphosphorylation, a key step in the aggregation cascade. Mechanistically, the protective effects of E2 were linked to the enhancement of protein quality control pathways; indeed, E2 treatment stimulated autophagic flux in both sALS lymphoblasts and NSC-34 cells and was associated with the activation and nuclear translocation of estrogen receptor α (ERα). Furthermore, in a cellular model expressing cytoplasm-restricted TDP-43 bearing mutations in the nuclear localization signal, E2 modulated the biophysical properties of TDP-43 inclusions, promoting the formation of smaller, more numerous, and more spherical assemblies. Fluorescence Recovery After Photobleaching (FRAP) analysis confirmed that E2 maintained these inclusions in a more dynamic, liquid-like state, counteracting their pathological maturation into less mobile, gel-like structures. Finally, in vitro biophysical assays using purified recombinant TDP-43 ruled out a direct action of E2 on modulating the protein phase behavior, suggesting that its effects are mediated through receptor-dependent mechanisms. In conclusion, this study demonstrates that estrogens could exert a pleiotropic, cell-mediated neuroprotective effect against TDP-43 mislocalization and assembly state. This protection does not result from direct binding of estrogens to TDP-43, but rather from the activation of ERα signaling and proteostasis networks, thereby providing a robust molecular rationale for the observed sex differences in ALS incidence and identifying the estrogen signaling pathway as a promising therapeutic target for mitigating neurodegeneration.