Neuronal hemoglobin induces α-synuclein cleavage and loss of dopaminergic neurons

Backgroud α-synuclein, a protein involved in the pathogenesis of several neurodegenerative disorders, is subjected to several post-translational modifications. Among them, C-terminal truncation seems to increase its aggregation propensity in vitro . Hemoglobin is the major protein in erythrocytes to carries oxygen and recently is found to be expressed in dopaminergic neurons and to be involved in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Methods To assess the role of hemoglobin in α-synuclein post-translational modification and in dopamine cells physiology, we over-expressed α and β-chains of Hb in iMN9D dopamine cells to evaluate its effect on α-synuclein truncation. Using an AAV9 we expressed α and β-chains of hemoglobin in dopamine neurons of Substantia Nigra pars compacta and evaluate its effect on α-synuclein post-translational modification, dopamine neurons survivals and behavioural outcome. Results The over-expression of α and β-chains of hemoglobin in iMN9D dopamine cells increased C-terminal truncation of α-synuclein when cells were treated with α-synuclein preformed fibrils. This cleavage was led at least in part by Calpain protease. Hemoglobin over-expression in Substantia Nigra pars compacta induced a similar pattern of α-synuclein truncation and a decrease in tyrosine hydroxylase expression, unveiling a decrease of dopamine neurons of about 50%. This dopamine cells loss led to a mild motor impairment and a deficit in recognition and spatial working memory. Conclusion Our study reveals a novel role for hemoglobin in α-synuclein post-translational modification and in dopamine neurons homeostasis suggesting neuronal hemoglobin is an important modifier in synucleinopathies such as Parkinson’s disease. ### Competing Interest Statement The authors have declared no competing interest. * α-syn : alpha-synuclein Ms : monomers PTMs : post-translational modifications pSer-129 : phosphorylated serine 129 ΔC- α-syn : C-terminal truncated α-syn FL- α-syn : full-length α-syn PD : Parkinson’s disease LBD : Lewy Body Dementia MSA : Multiple System Atrophy SNpc : Subtantia nigra pars compacta GCIs : Glial cytoplasmic inclusions DA : dopaminergic Hb : hemoglobin nHb : neuronal Hb MPP+ : 1-methyl-4-phenylpyridinium PFFs : pre-formed fibrils AFM : Atomic force microscopy NHP : non-human primates Capn I : Calpain I Ctsd : cathepsin D Cas1 : caspase 1 TH : tyrosine hydroxylase NOR : novel object recognition;