DOPA pheomelanin is increased in nigral neuromelanin of Parkinson’s disease and it exacerbates alpha-synuclein neurotoxicity

Objective Neuromelanin (NM) of the human substantia nigra (SN) has long been proposed as a key factor contributing to dopaminergic neuron vulnerability in Parkinson’s disease (PD). NM consists of pheomelanin and eumelanin moieties. Evidence supports that pheomelanin and eumelanin possess distinct chemical and biological characteristics. The present study aimed to investigate the relative composition and specific roles of pheomelanin and eumelanin moieties of NM in PD. Methods Pheomelanin and eumelanin components of NM in postmortem SN tissues from patients with PD were assessed by chemical degradation methods and compared with those from control subjects as well as patients with Alzheimer’s disease (AD). Additionally, synthetic pheomelanin and eumelanin were used to investigate their differential impacts on dopaminergic neuronal survival in a mouse model of PD overexpressing alpha-synuclein in the SN. Results We identified increased L-3,4-dihydroxyphenylalanine (DOPA) pheomelanin and increased ratios of dopamine (DA) pheomelanin markers to DA in PD SN compared to the controls. Eumelanins derived from both DOPA and DA were reduced in PD group. Melanin markers were unaltered in AD SN compared to the controls. Furthermore, we showed exacerbated dopaminergic neurodegeneration by synthetic DOPA pheomelanin and attenuated DA deficit by synthetic DOPA eumelanin in an alpha-synuclein mouse model of PD. Conclusion Our study provides insights into the different roles of pheomelanin and eumelanin moieties in PD pathophysiology. It forms a foundation for further investigations on pheomelanin and eumelanin individually as biomarkers and therapeutic targets for PD. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The study is funded by the joint efforts of The Michael J. Fox Foundation for Parkinson's Research (MJFF) and the ASAP initiative. MJFF administers the grant [ASAP-00312] on behalf of ASAP and itself. For the purpose of open access, the authors have applied a CC-BY public copyright license to the Author Accepted Manuscript (AAM) version arising from this submission. This work was also supported by NIH grants R01NS102735, the Milstein Medical Asian American Partnership Foundation 2015, the Farmer Family Foundation Initiative for Parkinson's Disease Research, and the Grigioni Foundation for Parkinson's Disease (Milan, Italy). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The ethics committee/IRB of Massachus Massachusetts General Hospital waived ethical approval for this work/ Not Human Subject Research Determination I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript. * AAV : Adeno-associated virus AD : Alzheimer’s disease 3-AHP : 3-Amino-4-hydroxyphenylalanine 4-AHP : 4-Amino-3-hydroxyphenylalanine 3-AHPEA : 3-Amino-4-ydroxyphenylethylamine 4-AHPEA : 4-Amino-3-hydroxyphenylethylamine AHPO : Alkaline hydrogen peroxide oxidation BT : Benzothiazine BZ : Benzothiazole CON : Control Cys : Cysteine Cys-DA : Cysteinyldopamine Cys-DOPA : Cysteinyldopa DA : Dopamine DOPA : L-3,4-dihydroxyphenylalanine DOPAC : 3,4-dihydroxyphenylacetic acid ECD : Electrochemical detection HPLC : High performance liquid chromatography HI : Hydroiodic acid Iba1 : Ionized calcium binding adapter molecule 1 NM : Neuromelanin p-αSyn : Phosphorylated alpha-synuclein PD : Parkinson’s disease PDCA : Pyrrole-2,3-dicarboxylic acid PMI : Postmortem interval PTCA : Pyrrole-2,3,5-tricarboxylic acid SN : Substantia nigra SNpc : SN pars compacta αSyn : alpha-Synuclein TH : Tyrosine hydroxylase TTCA : Thiazole-2,3,5-tricarboxylic acid UVD : UV detection.