Neuronal hemoglobin: new insights into the mechanism of ⍺-synuclein pathogenicity

α-synucleinopathies are a subset of progressive neurodegenerative disorders that share several features and, above all, α-synuclein (α-syn) accumulation. Lewy bodies and neurites are typical of Parkinson’s disease (PD) and dementia with Lewy bodies, while Multiple System Atrophy is characterized by the presence of glial cytoplasmic inclusions. Several mechanisms are involved in the pathogenesis of these diseases, ranging from genetic factors, environment, cellular disfunctions to α-syn misfolding. An increasing number of studies reported that α-syn is subjected to post-translational modifications within Lewy bodies. Among them, C-terminal truncation seems to increase its aggregation propensity. Hemoglobin (Hb) genes expression was recently demonstrated in the central nervous system and evidences suggest a putative role as oxygen reservoir and in mitochondrial respiration. Dysregulation of Hb expression has been associated to several neurodegenerative disorders and, in particular, to PD. Previous data from the laboratory of Prof. Gustincich showed that Hb alters mitochondrial genes expression1, confers dopaminergic (DA) cells’ susceptibility to MPP+ and rotenone in vitro and impairs performance associated to motor learning in vivo2. Interestingly, the presence of Hb - α-syn complexes in the brain of aging cynomolgus monkeys has recently been demonstrated3. In this study, we aimed to unveil the interplay between Hb and α-syn and its contribution to synucleinopathies. To this purpose, we took advantage of MN9D-Nurr1Tet-On dopaminergic neuroblastoma cell lines (iMN9D) stably transfected with α and β chains of Hb1 and α-syn preformed fibrils (PFFs) as a model of α-syn pathogenicity. We first demonstrate that iMN9D cells internalize PFFs and are therefore a suitable system to study α-syn pathogenicity. Then, we show that PFFs treatment is toxic to cells and leads to the accumulation of α-syn C-terminal truncated species (DC-α-syn) triggered in the presence of Hb. Given the relevance of C-terminal cleavage products in terms of α-syn aggregation and toxicity, we investigate the intracellular proteases likely involved in α-syn truncation. Among them, we provide evidence that, in our model, calpains are involved in α-syn truncation. In conclusion, taken together, these findings strongly support Hb implication in α-syn processing and, therefore it exerts a significant role in the pathogenesis of α-synucleinopathies.