Neuromelanin organelles are specialized autolysosomes that accumulate undegraded proteins and lipids in aging human brain and are likely involved in Parkinson's disease

Abstract

During aging, neuronal organelles filled with neuromelanin (a dark-brown pigment) and lipid bodies accumulate in the brain, particularly in the substantia nigra, a region targeted in Parkinson's disease. We have investigated protein and lipid systems involved in the formation of these organelles and in the synthesis of the neuromelanin of human substantia nigra. Membrane and matrix proteins characteristic of lysosomes were found in neuromelanin-containing organelles at a lower number than in typical lysosomes, indicating a reduced enzymatic activity and likely impaired capacity for lysosomal and autophagosomal fusion. The presence of proteins involved in lipid transport may explain the accumulation of lipid bodies in the organelle and the lipid component in neuromelanin structure. The major lipids observed in lipid bodies of the organelle are dolichols with lower amounts of other lipids. Proteins of aggregation and degradation pathways were present, suggesting a role for accumulation by this organelle when the ubiquitin-proteasome system is inadequate. The presence of proteins associated with aging and storage diseases may reflect impaired autophagic degradation or impaired function of lysosomal enzymes. The identification of typical autophagy proteins and double membranes demonstrates the organelle's autophagic nature and indicates that it has engulfed neuromelanin precursors from the cytosol. Based on these data, it appears that the neuromelanin-containing organelle has a very slow turnover during the life of a neuron and represents an intracellular compartment of final destination for numerous molecules not degraded by other systems.

Fig. 1

Transmission electron microscopy images of NM-containing organelles in human SN tissue (a–c) and after the isolation procedure (d). a–c SN tissue of 89 y.o. healthy subject. Intraneuronal NM-containing organelles of the SN are membrane bounded (black arrowhead in a and b) and contain large amounts of NM pigment (black and electron dense), a protein matrix and lipid bodies (asterisk). Scale bar =1 µm in a. Large lipid bodies (asterisk) are surrounded by a membrane as demonstrated in b (arrow), although the images do not distinguish between a bilayer and single layer membrane. Considering that brain samples used in this study were post mortem tissues, it is striking that there is often a double membrane around many of the organelles. At higher magnification, a double membrane delimiting NM-containing organelle is clearly visible (empty arrowhead in c). d NM-containing organelles isolated from the SN tissue of 89 y.o. healthy subject (the same subject of a–c). The purity and integrity of isolated NM-containing organelles is clearly demonstrated by transmission electron microscopy: low magnification d demonstrates that cellular and subcellular debris are completely absent. The outer limiting membrane is not apparent, but the constituents of the organelles appear intact with NM pigment, many lipid bodies (asterisks) and membranes of lipid bodies. Scale bar = 1 µm in d

Fig. 2

Histogram of cellular distribution of the 293 representative proteins found in all analyzed samples shown as relative number of SpC vs. cellular compartments. For details of subjects and preparation of samples for LC-MS analysis of proteins, see Methods. Some proteins may have multiple cellular locations: for each protein the most typical and representative cellular location was assigned. The different types of samples are represented by different colors (ORG, TIS-NM, and ORG-NM) and gray bars refer to overall representative proteins considered as a single data set (indicated with “All Samples”). The “Rel. # SpC (%)” is the total number of SpC for a specific class of proteins (i.e., lysosomal) referred to the overall number of SpC of representative proteins in each sample: this value represents the relative abundance of a particular class of proteins in one sample (see also Supplementary Table 1). The term “Vesicles” refers to vesicle trafficking, including proteins involved in vesicular transport, fusion, etc. The category “Unknown cell location” consists of proteins for which a cellular location was still unclear, while the class “Uncharacterized proteins” comprises proteins for which, at the moment of data analyses, a complete characterization and/or role was missing

Fig. 3

Area-proportional Euler diagram of the 293 representative proteins (detected by SpC ≥ 2 as average value in at least one of the three types of samples) identified in ORG, TIS-NM, or ORG-NM. For details of subjects and preparation of samples for LC-MS analysis of proteins, see Methods. The diagram was calculated using the EulerAPE tool (Methods) and by using NCBI accession (GI number). Outside the diagram we report for each type of sample the following values: (i) in brackets, the number of proteins detected as representative (with SpC ≥ 2) uniquely in that type of sample, as reported in Table 1; (ii) without brackets, the number of representative proteins plus those identified as non-representative (with SpC < 2) in that specific sample but listed as representative (with SpC ≥ 2) in at least one of the other type of samples (e.g., a protein that was detected as non-representative in ORG sample but as representative in TIS-NM would be included in the count for ORG). Numbers in non-overlapping areas of circles report the representative proteins found uniquely in that type of sample. The overlapping areas correspond to proteins shared by two or three different types of samples: e.g., a protein detected in all samples but as representative only in ORG would be included in the overlapping area of 35 proteins shared between ORG, TIS-NM, and ORG-NM. Percentages in parentheses represent the ratio between the total number of SpC of proteins belonging to one area of the diagram and the overall number of SpC of representative proteins detected in all samples. The highest percentage value is located in the area shared between all three types of samples. The detailed list of proteins is reported in Supplementary Data 1

Fig. 4

IEM of SN from healthy aged subjects for selected proteins. For number of IEM experiments, see Methods. CTSD (73 y.o.; gold particles = 20 nm). LAMP2 (86 y.o.; gold particles = 20 nm). MAP1LC3B (69 y.o.; gold particles = 15 nm). SCARB2 (69 y.o.; gold particles = 15 nm). SNCA (63 y.o.; gold particles = 15 nm). UBA52 (63 y.o.; gold particles = 15 nm). Lipid bodies are indicated by asterisks. NM pigment of the NM-containing organelles appears as black and electron dense granular aggregates. Scale bar in each panel = 250 nm

Fig. 5

WB (for proteins detected by IEM in Fig. 4) performed on SN tissue lysates and on ORG samples. For number of WB analyses, see Methods. CTSD (protein content ratio SN tissue lysate/ORG = 3.1). The band present in both SN tissue lysate (16 pooled tissues, from 48 to 85 years of age) and ORG sample (isolated from one subject, 81 y.o.) corresponds to the mature CTSD heavy chain which is highly enriched in ORG sample, considering that the total protein content in ORG was 3.1-fold lower than that of SN tissue lysate. LAMP2 (protein content ratio SN tissue lysate/ORG = 1.0). LAMP2 protein was lightly present in ORG sample (isolated from one subject, 83 y.o.), while in SN tissue lysate (13 pooled tissues, from 62 to 86 years of age) this protein is largely expressed. The antibody used here recognizes all three LAMP2 isoforms. MAP1LC3B (protein content ratio SN tissue lysate/ORG = 1.8). The black arrowhead indicates the MAP1LC3B-I form, which was more prevalent in SN tissue lysate (five pooled tissues, from 73 to 85 years of age) than the MAP1LC3B-II form (empty arrowhead indicating the phosphatidylethanolamine conjugated form). In ORG sample (isolated from one subject, 77 y.o.), the MAP1LC3B-I form was abundant while MAP1LC3B-II form was undetectable. SCARB2 (protein content ratio SN tissue lysate/ORG = 2.3). Here we note an enrichment of SCARB2 in ORG sample (isolated from one subject, 77 y.o.) if compared to SN tissue lysate (five pooled tissues, from 73 to 85 years of age), considering that the total protein content in ORG was 2.3-fold lower than that of SN tissue lysate. SNCA (protein content ratio SN tissue lysate/ORG = 3.4). The black arrowhead indicates the soluble-monomeric form of SNCA which is clearly visible in SN tissue lysate (nine pooled tissues, from 67 to 85 years of age) while undetectable in ORG sample (isolated from one subject, 66 y.o.). Other bands at higher molecular weight are present in SN tissue lysate, corresponding to fibrils and aggregates with possible modifications. In the ORG sample, two main bands are clearly visible corresponding to some aggregated/modified forms of SNCA (at ~50 and ~58 kDa) which are present also in SN tissue lysate. UBA52 (protein content ratio SN tissue lysate/ORG = 2.7). The black arrowhead indicates the free ubiquitin that is scarcely visible in SN tissue lysate (eight pooled tissues, from 62 to 89 years of age), but abundant in the ORG sample (isolated from one subject, 66 y.o.). The WB also reveals the presence of large number of immunoreactive high molecular weight bands corresponding to high amounts of poly-ubiquitinated proteins, both in SN tissue lysate and highly enriched in the ORG sample, although the total protein content in ORG was 2.7-fold lower than that of SN tissue lysate

Fig. 6

LC-MS analysis of lipids isolated from TIS-NM and ORG samples. The TIS-NM sample here represented was isolated from a pool of seven subjects (from 71 to 85 years of age), while the ORG sample was isolated from two pooled subjects (74 and 89 y.o.). Mass spectra (averaged mass spectra, range 1200–1500 m/z) demonstrate the presence of dolichols species in both samples. We highlight the series of singly charged ions with different chain lengths, corresponding to dolichols with terminal hydroxyl group, their oxidized derivative dolichoic acids, and acetate adducts of dolichols species. Both spectra selectively show dolichol species with chain lengths ranging from 17 to 21 isoprene units, although few dolichol species with lower and higher number of isoprene units were found in lipid extracts from both samples (Results). Abbreviations used in the figure: Dol, dolichol; Dol-Ac, dolichol acetate; Dol-CA, dolichoic acid

Fig. 7

High performance TLC analysis of total lipid extracts obtained from TIS-NM and ORG samples. The TIS-NM sample here represented was isolated from a pool of four subjects (from 62 to 86 years of age), while lipids of from three ORG samples (each isolated from three different subjects, respectively 62, 61 and 77 y.o.) were pooled before loading onto the TLC plates. After separation, lipids were detected by spraying the TLC plate with anisaldehyde. In TIS-NM sample the intense spot at the solvent front likely corresponds to dolichols and dolichoic acids, as confirmed by LC-MS (Fig. 6). The content of sphingomyelin, galactosylceramide, sulfatides (typical myelin lipids), and lactosylceramide is higher than phosphatidylethanolamine and phosphatidylcholine (glycerophospholipids). In the ORG samples, the main components are again dolichols and dolichoic acids at the solvent front. In this sample there are comparable amounts of sphingolipids (lactosylceramide and sphingomyelin) and glycerophospholipids. The arrows at the margin of the image indicate the position of pure standard lipids co-chromatographed with the samples, as described in Methods. Abbreviations used in the figure: GalCer, galactosylceramide; GD1a, GD1b, GM1, GT1b, gangliosides GD1a, GD1b, GM1, GT1b; GlcCer, glucosylceramide; LacCer, lactosylceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; ST, sulfatides

Fig. 8

Hypothesized scheme summarizing NM-containing organelle formation in human SN. a, b In the cytosol of SN neurons, DA can be oxidized to semiquinones and quinones via iron catalysis, and these highly reactive compounds can react with aggregated and β-structured proteins that accumulate in the cytosol. c The oxidative polymerization of quinones initiates with the formation of the melanin-protein complex which can also bind high levels of metals, especially iron. During this step drugs and toxicants can also bind to the melanin-protein complex. Proteins damaged by misfolding and DA-adducts formation may be recognized and bound by ubiquitins (green) and alpha-crystallin B chain, in the attempt to degrade damaged and/or misfolded proteins in the proteasome pathway. It may be that ubiquitinated-NM-derived products are too large and damaged to be degraded by the proteasome system. d, e The resulting undegradable material accumulates in the cytosol. GPNMB and tubulin polymerization-promoting protein could be engaged at this step, since these proteins are involved in the formation of aggresome-like structure and degradation of cellular debris.^, The accumulated undegradable material is then taken up into autophagic vacuoles by the phagophore, an isolation double membrane which engulfs bulk material for macroautophagy. This is confirmed by the presence of some typical macroautophagic markers such as MAP1LC3B (red) and SQSTM1 (orange), and by the presence of a double membrane surrounding the NM-containing organelle as displayed by electron microscopy (Fig. 1c). f These autophagic vacuoles fuse with lysosomes, shown in the scheme with numerous enzymes (black), different membrane proteins (blue) and the proton pumps (violet), to become autolysosomes containing the enzymes, proteins and lipids of lysosomes. After fusion with lysosomes, the undegraded and NM-derived material contained in the autophagic vacuoles can interact with other lipids and other proteins carried by lysosomes. A decreased lysosomal enzyme efficiency and reduced fusion capacity could occur also as a consequence of aging, oxidative stress and NM accumulation. g These organelles can fuse with other vesicles or with other autophagic vacuoles containing NM precursors or with old NM-containing organelles, etc. This fusion could lead to the accumulation in the lumen of the organelle of membranous portions which would otherwise be degraded, while dolichols in particular are not chemically decomposed and accumulate with other undegraded lipids leading to the formation of lipid bodies. Dolichols (or dolichyl esters) may be transported into the organelle by vesicle transport and membrane fusion. Fusion of organelles could be mediated by RAB5A, tubulin polymerization-promoting protein, microtubule-associated protein tau and other related proteins. This aged organelle, due to its particular content of undegradable NM pigment together with damaged and oxidized proteins, lipids and metals, is a reservoir of buffered toxins (red stars). Under conditions of cellular damage these toxins together with NM pigment could be released and induce neuroinflammation and neurodegeneration. h The final NM-containing organelle is the result of a complex and continuous process occurring during aging, that leads to the accumulation of undegradable material in specialized pigmented “autophagic lysosomes”. Figure modified from previously published papers, by permission of Springer and by permission of Elsevier