Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra (SN) pars compacta. NMGs are of high clinical relevance, because neurons with a high NMG content are more vulnerable in the context of Parkinson"s disease (PD) and dementia with Lewy bodies (DLB). Nevertheless, the role of NMGs in health and disease remains incompletely understood. Since NMGs are absent in the brain of most common laboratory animals without genetic modifications, researchers are dependent on human post-mortem brain tissue, which is highly requested and therefore limited.

To overcome this limitation, we established a protocol for the proteomic characterization of NMGs in comparison to the surrounding SN (SNSurr.) tissue, based on the use of laser microdissection and bottom-up proteomics (Wulf et al., 2021). Two follow-up studies have been conducted using this workflow, with the proteome of NMGs being characterized under physiological conditions (Wulf et al., 2022a) and neurodegenerative conditions using post-mortem brain tissue of DLB cases (Wulf et al., 2022b). A comparison of the proteomic profiles of NMGs from CTRL and DLB cases revealed that alpha-synuclein and protein S100A9 were significantly higher abundant in NMGs of DLB cases, indicating the presence of a DLB-specific proteomic profile in NMGs.

In the current study, we analyzed post-mortem brain tissue from eight PD and ten CTRL cases and compared the proteomic content of NMG and SNSurr. samples. In concordance to our study on DLB-specific proteomic changes, alpha-synuclein was again significantly higher abundant in PD NMGs compared to CTRL NMGs. Despite this concordance, other upregulated proteins differed, indicating that proteins potentially co-accumulating in NMGs with alpha-synuclein may be disease-specific and thus indicate disease specific pathomechanisms.

From a methodological perspective, we were able to drastically reduce the amount of tissue required and the analysis time after laser microdissection. By using the EvoSep One in combination with the TimsTOF Pro 2, we were able to reach yet unprecedentedly high numbers in protein identifications while reducing the sample input more than 10-fold. These results drastically demonstrate the benefits of state-of-the-art instrumentation for the research on NMGs.