High-Resolution Elemental Analysis of Neuromelanin-Containing Organelles in Human Locus Coeruleus.

Abstract

Neuromelanin-(NM) containing organelles are sub-cellular auto-lysosomal structures composed of three main compartments: NM pigment, protein matrix, and lipid bodies. These organelles accumulate during aging and are found predominantly in the catecholaminergic neurons of Substantia Nigra (SN) and Locus Coeruleus (LC), the main regions affected in Parkinson's disease (PD). NM serves a protective function by sequestering potentially toxic metals like Cu, Fe, and Al. However, NM released from degenerating neurons may lead to a cascade of events resulting in neuroinflammation and neurodegeneration. Therefore, elemental analysis of NM-containing organelles presents a crucial step to understand aging and PD. In LC, such studies are limited because NM isolation requires large postmortem cohorts and analyses may be impaired by tissue processing. By integrating high resolution electron microscopy (EM), nano-secondary ion mass spectrometry (nano-SIMS), and energy dispersive X-ray (EDX) microscpectroscopy, the elemental composition of intact NM-containing organelles was analyzed in seven postmortem LC tissues. Chemical mapping with down to 5-10 nm lateral resolution (EDX) fostered discrimination of structural composition (N, P, S, Cl) and metal storage (Al, Ca, Fe) across neurons and within individual NM-containing organelle sub-compartments (with diameters down to 0.2 μm for lipid bodies) from the same sample. NM-containing organelles were identified by an elemental fingerprint pattern. Metals accumulations were localized predominantly to the NM pigment compartment identified by its pheomelanin-rich portion (S). This confirms NM's role in accumulating physiological as well as potentially toxic metal species. Moreover, semi-quantitative analyses provided insights into inter- and intra-subject NM metal accumulation, showing that S and Fe exhibited a positive aging trend. Hemispheric asymmetry was observed for Al, Ca, and Fe, with higher levels observed in NMs of the right brain hemisphere suggesting region-specific accumulation that warrants further investigation to better understand aging-related changes and the neuronal vulnerability of the LC in PD.