cells Article Proteomic Characterization of Synaptosomes from Human Substantia Nigra Indicates Altered Mitochondrial Translation in Parkinson’s Disease 1, 1,2, 1 1,2 3 Sarah Plum y, Britta Eggers y , Stefan Helling , Markus Stepath , Carsten Theiss , Renata E. P. Leite 4,5, Mariana Molina 4, Lea T. Grinberg 4,6, Peter Riederer 7,8, Manfred Gerlach 9, 1,2, 1,2, , Caroline May z and Katrin Marcus * z 1 Medizinisches Proteom-Center, Medical Faculty, Ruhr-University Bochum, 44801 Bochum, Germany; [email protected] (S.P.); [email protected] (B.E.); [email protected] (S.H.); [email protected] (M.S.); [email protected] (C.M.) 2 Medical Proteome Analysis, Center for Proteindiagnostics (PRODI), Ruhr-University Bochum, 44801 Bochum, Germany 3 Department of Cytology, Institute of Anatomy, Ruhr-University Bochum, 44780 Bochum, Germany; [email protected] 4 Department of Pathology, LIM22, University of Sao Paulo Medical School, Sao Paulo 01246-903, Brazil; [email protected] (R.E.P.L.); [email protected] (M.M.); [email protected] (L.T.G.) 5 Division of Geriatrics, LIM 66, University of Sao Paulo Medical School, Sao Paulo 01246-903, Brazil 6 Department of Neurology, Memory and Aging Center, University of California, San Francisco, CA 94158, USA 7 Center of Mental Health, Clinic and Policlinic for Psychiatry, Psychosomatics and Psychotherapy, University Hospital Wuerzburg, Margarete-Höppel-Platz 1, 97080 Wuerzburg, Germany; [email protected] 8 Psychiatry Department of Clinical Research, University of Southern Denmark Odense University Hospital, Winslows Vey 18, 5000 Odense, Denmark 9 Center of Mental Health, Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, University Hospital of Wuerzburg, University of Wuerzburg, 97080 Wuerzburg, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-234-32-18106 These authors contributed equally to this work. y These authors contributed equally to this work. z Received: 15 September 2020; Accepted: 24 November 2020; Published: 2 December 2020 Abstract: The pathological hallmark of Parkinson’s disease (PD) is the loss of neuromelanin-containing dopaminergic neurons within the substantia nigra pars compacta (SNpc). Additionally, numerous studies indicate an altered synaptic function during disease progression. To gain new insights into the molecular processes underlying the alteration of synaptic function in PD, a proteomic study was performed. Therefore, synaptosomes were isolated by density gradient centrifugation from SNpc tissue of individuals at advanced PD stages (N = 5) as well as control subjects free of pathology (N = 5) followed by mass spectrometry-based analysis. In total, 362 proteins were identified and assigned to the synaptosomal core proteome. This core proteome comprised all proteins expressed within the synapses without regard to data analysis software, gender, age, or disease. The differential analysis between control subjects and PD cases revealed that CD9 antigen was overrepresented and fourteen proteins, among them Thymidine kinase 2 (TK2), mitochondrial, 39S ribosomal protein L37, neurolysin, and Methionine-tRNA ligase (MARS2) were underrepresented in PD suggesting an alteration in mitochondrial translation within synaptosomes. Cells 2020, 9, 2580; doi:10.3390/cells9122580 www.mdpi.com/journal/cells Cells 2020, 9, 2580 2 of 19 Keywords: synaptosomes; proteomics; Parkinson’s disease; substantia nigra pars compacta; mitochondrial pathology; mitochondrial translation 1. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease in the elderly [1]. Lewy body disease, the neuropathological counterpart of PD, features a preferential loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta (SNpc), accompanied by the accumulation of intracellular proteinaceous inclusions named Lewy bodies and a reduction in striatal dopamine as one of its most striking findings [2]. This ongoing loss of dopaminergic neurons mainly leads to clinical diagnosis due to occurrence of motor symptoms such as rigidity, tremor and bradykinesia, which results from a reduction of about 70% of striatal dopamine [3,4]. However, neuronal loss in PD occurs in many other brain regions, including the locus coeruleus, nucleus basalis of Meynert, pedunculopontine nucleus, raphe nucleus, dorsal motor nucleus of vagus, amygdala, and hypothalamus [5]. Neurodegeneration of these neurons results in the dysfunction of other neurotransmitter systems including cholinergic, GABAergic, glutamatergic and noradrenergic neurotransmission [6]. That leads to a wide range of non-motor symptoms that sometimes even precede the typical movement disorder, such as olfactory dysfunction, sleep disturbances (i.e., rapid eye movement sleep behavior disorder), psychiatric symptoms such as depression, autonomic dysfunction, pain, and fatigue [7]. The causes of these neuronal losses have hardly been investigated so far. Many of these neurons project into the SN and are thus probably involved in the neurodegenerative processes there. Synaptosomes represent synapses detached during tissue lysis from neuronal cell bodies [8,9] and still contain the machinery required for signal transduction [10]. The study of synaptosomes provides insights into the physiological and pathophysiological processes in neurotransmission and synaptic protein–protein interaction networks [11]. Differences in the protein composition of synaptosomes in the context of a disease might indicate a disturbed signal transduction in the synapses. Pathological deficits in the synaptic processes in neurological disorders have been studied in the past especially for Alzheimer’s [12–15], Huntington’s disease [16], Lewy body disease [17] and PD [18] as well as schizophrenia [19–21]. Omics approaches including proteomics are promising new avenues to study synaptosomes [22,23]. Indeed, human synaptosomes have so far only been investigated in a handful of proteomics studies [24,25] mainly in Alzheimer’s disease [10,26,27], schizophrenia [28,29] and only one investigates pathophysiological molecular processes in Lewy body disease (LBD) [30]. To elucidate a possible pathophysiological effect leading to PD caused by a loss of neurons that project into the SN, we analyzed the proteome of synaptosomes from human postmortem SNpc tissue. In the past, different protocols for the enrichment of synaptosomes by, e.g., density gradient centrifugation, have been established [31] (for review see [32]). Here, a combined approach of a protocol designed by Dunkley et al. [33] with a strategy to enrich neuromelanin granules allowing for a case-specific enrichment [34] was used. Differences in the synaptosomal proteome of PD subjects and controls were identified by bottom-up liquid chromatography tandem mass spectrometry (LC-MS/MS). From these results, a synaptosomal core proteome of the SNpc could be derived for the first time. Disease-related differences were detected, indicating an altered mitochondrial translation in synaptosomes of individuals suffering from PD. Cells 2020, 9, 2580 3 of 19 2. Materials and Methods 2.1. Ethical Statement Human postmortem SNpc tissues were provided by the German Brain Bank in Wuerzburg, Germany, and the Brazilian Brain Bank in São Paulo, Brazil. The use of human brain tissue was approved by the ethics committee of the University Clinics of Wuerzburg, Germany (file number 78/99), the ethics committee of the Ruhr-University Bochum, Germany (file number 4760-13), the ethics committee of the University of São Paulo (file number 361/10), and the Brazilian national health ministry (file number 16380). 2.2. Study Cohort for Subsequent Proteomic Analyses Details about the analyzed study groups are shown in Table1. For all PD cases, the neuropathological severity of the disease was assessed according to measures defined by Braak et al. and ranged between stages 3 to 6 [35]. Table 1. Characteristics of the study groups. PD Tissue Age Ø Age PMI Ø PMI (h) Braak/ Group Gender DS Weight CDR CERAD (in years) SD ± (h) SD AD (g) ± Braak Female 88 NA ~13:00 0.2306 NA NA NA Male 80 NA 19:39 0.4616 NA NA NA Control Female 73 78.8 6.9 NA 13:3014:04 0.13 0.4076 NA NA NA ± ± Male 82 NA 12:11 0.2530 NA NA NA Female 71 NA 12:00 0.3925 NA NA NA Female 87 3 13:08 0.2461 3/3 0 none Male 88 5 11:20 0.3607 5/3 0.5 moderate PD Female 7580.8 6.8 3 14:4014:19 0.12 0.6556 3/2 0 moderate ± ± Male 81 6 19:10 1.1524 6/2 3 moderate Female 73 3 13:20 0.4838 3/2 0 none AD: Alzheimer’s disease; Ø Age SD: average age and standard deviation; DS: disease stage according to neuropathological classification (Braak± scale); PMI: postmortem interval; Ø PMI SD: average PMI and standard deviation; NA: not applicable, CDR: clinical dementia rating, CERAD: Consortium± to Establish a Registry for Alzheimer’s Disease. 2.3. Tissue and Sample Preparation for LC-MS/MS Experiments All subjects died of natural causes, and their brains were dissected and stored at 80 C, − ◦ as previously described [36]. A detailed description of the respective procedures can be found elsewhere [37]. Brain tissue was obtained within 24 h of death. One hemisphere was fixed in 4% buffered paraformaldehyde, and selected brain areas from the other hemisphere were frozen at 80 C. Internationally accepted neuropathological criteria were used to stage and diagnose the brain − ◦ pathologies [35,38–41]. The neuropathological term Lewy body disease (LBD) was used for all diseases associated with Lewy bodies, thereby eliminating the distinction between PD, PDD (Parkinson’s disease dementia) and DLB (dementia with lewy bodies). We staged LBD using the criteria of Braak and colleagues. For sample characteristics, see Table1. Synaptosomes were enriched from PD and control SNpc tissues (each N = 5) with centrifugation as described [34]. Pelleted synaptosomes were dissolved and lysed by addition of 20 µL deionized water, resuspension with a pipette five times, and then vortexed. Protein concentration was determined by amino acid analysis [34]. Then, 5 µg of each sample was diluted with deionized water to an end volume of 14 µL.