Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles

Abstract

Melanin, the pigment in hair, skin, eyes, and feathers, protects external tissue from damage by UV light. In contrast, neuromelanin (NM) is found in deep brain regions, specifically in loci that degenerate in Parkinson's disease. Although this distribution suggests a role for NM in Parkinson's disease neurodegeneration, the biosynthesis and function of NM have eluded characterization because of lack of an experimental system. We induced NM in rat substantia nigra and PC12 cell cultures by exposure to l-dihydroxyphenylalanine, which is rapidly converted to dopamine (DA) in the cytosol. This pigment was identical to human NM as assessed by paramagnetic resonance and was localized in double membrane autophagic vacuoles identical to NM granules of human substantia nigra. NM synthesis was abolished by adenoviral-mediated overexpression of the synaptic vesicle catecholamine transporter VMAT2, which decreases cytosolic DA by increasing vesicular accumulation of neurotransmitter. The NM is in a stable complex with ferric iron, and NM synthesis was inhibited by the iron chelator desferrioxamine, indicating that cytosolic DA and dihydroxyphenylalanine are oxidized by iron-mediated catalysis to membrane-impermeant quinones and semiquinones. NM synthesis thus results from excess cytosolic catecholamines not accumulated into synaptic vesicles. The permanent accumulation of excess catechols, quinones, and catechol adducts into a membrane-impermeant substance trapped in organelles may provide an antioxidant mechanism for catecholamine neurons. However, NM in organelles associated with secretory pathways may interfere with signaling, as it delays stimulated neurite outgrowth in PC12 cells.

Figure 1

Neuromelanin in cultured neurons. (a) Bright-field image of living SNC neurons exposed to vehicle and photographed 3 weeks postplating. In these control cultures, no pigment was observed. Arrows indicate examples of cell bodies. (b) Bright-field image of living SNC neurons derived from the same dissection exposed to 50 μM l-DOPA (11 days). Dark brown NM granules are distributed in a pattern identical to that in SNC neurons in human brain. The granule distribution was primarily in cell bodies. The double-headed arrow indicates the most highly NM-expressing cell in the field. Less pigmented cells are indicated with single-headed arrows. (Scale bar = 5 μm.) (c) A higher-magnification bright-field image of NM in living SNC culture after exposure to 50 μM l-DOPA (14 days). Arrows indicate samples of NM granules that are in focus in this plane. (Scale bar = 5 μm.) (d) NM granules in a cultured SN neuron exposed to 50 μM l-DOPA (14 days). The white arrows indicate individual NM granules. The granules are essentially identical to those in human SN, although there is little or no lipofuscin. Sections are stained with osmium and uranyl acetate only. (Scale bar = 1 μm.) Controls do not display NM. (e) At higher power, a double membrane is discerned (arrows) as in NM granules in vivo. (Scale bar = 200 nm.)

Figure 2

Human NM granules. NM granules from human SN. The arrows indicate the double membranes. ∗ indicates lipofuscin deposits within the granules. (Scale bar = 100 nm.)

Figure 3

(a) Expression of NM granules. Granules were counted in SN cultures and scored as displaying 0, 1–5, 5–10, or >10 pigmented granules per neuron (mean ± SEM, four culture dishes per condition, = 100 cells rated per culture dish). The cultures were derived from the same batch of SNC dissections and were exposed to vehicle, l-DOPA, or l-DOPA with l-NAC once, and fixed (so that the age would be identical for all groups) 26 days after exposure. The data are displayed as fraction of neurons within each category (i.e., n of neurons in a bin of #granules/N of total neurons in that treatment). There was an increase in pigmented granules for l-DOPA (50 μM) over controls (P < 0.001, χ² test) and l-DOPA with l-NAC (500 μM) over controls (P < 0.001, χ² test). The total number of surviving neurons was not different between the three conditions. l-NAC-only treated cultures showed no pigmented granules. (b) Effect of VMAT2 overexpression and DES. Cultures derived from the same batch of SNC dissections were induced to overexpress VMAT2 by exposure to an adenoviral vector. Control cultures were exposed to a similar adenovirus construct that lacked the VMAT2 sequence. Cultures then were exposed to vehicle or l-DOPA and fixed 7 days after exposure. There was an increase in pigmented granules for l-DOPA (50 μM) over controls (P < 0.001, χ² test). VMAT2 overexpressing cultures exposed to l-DOPA displayed fewer pigmented granules than l-DOPA alone (P < 0.001, χ² test in both this trial and an independent experiment where cultures were exposed to l-DOPA for 14 days). Cultures that overexpressed VMAT2 but were not exposed to l-DOPA showed no pigmented granules (not shown). Cells exposed to both l-DOPA and DES (10 μM) displayed fewer pigmented granules than l-DOPA alone (P < 0.001, χ² test in two separate trials). DES-only treated cultures showed no pigmented granules (not shown).

Figure 4

NM in PC12 cells. (a) In control PC12 cells, the small electron dense bodies (single-headed arrows) are normal secretory dense core granules. (b) In cultures exposed to 100 μM d-DOPA for 60 days (NGF added on day 50), both dense core granules (single-headed arrows) and larger NM granules (double-headed arrows) are observed. A double membrane is indicated by arrowheads. ∗ indicates a classic autophagic vacuole. (Scale bar = 200 nm.)

Figure 5

EPR of cell culture and human NM. (a) EPR spectra of control PC12 cells exposed to vehicle for 3 months; PC12 cells derived from the same parent culture exposed to 100 μM d-DOPA for 3 months; isolated NM from PC12 cells after EDTA treatment, which removes the peak corresponding to iron radical; and isolated NM from human SNC. The two peaks in human NM are at identical positions to the major peaks in PC12 cells. (b) Amplitudes of the semiquinone peaks for the control PC12 cultures and the cultures exposed to d-DOPA. The bars indicate the mean amplitude ± SEM (three readings) from cells derived from the same parent culture.

Figure 6

Conditions that promote NM synthesis delay neurite outgrowth. Cells were scored as displaying processes if they exhibited a neurite that extended at least one cell body length. Cells were exposed to 100 μM d-DOPA for 30 days, with replacement of medium containing the compound in 2- to 3-day intervals. After two additional replacements with medium without d-DOPA and subsequent washing, NGF (50 mg/ml) was added. The cells were scored at 1- to 2-day intervals for neurite outgrowth (n = 3 cultures, 100 cells scored per culture, ± SEM). Similar results were observed in five independent experiments and after similar l-DOPA exposure (not shown).

Figure 7

Proposed model for NM synthesis in SN DA neurons. 1) l-DOPA is taken up by a plasma membrane amino acid transporter. 2) l-DOPA is synthesized endogenously by tyrosine hydroxylase (TH). 3) DA is produced by aromatic acid decarboxylase (AADC). 4) Additional cytosolic DA is by the DA uptake transporter. 5) Synaptic vesicles (SV) and endosomes (not shown) accumulate cytosolic DA via VMAT2. 6) Cytosolic DA is metabolized in mitochondria (Mito) via monoamine oxidase. 7) Excess cytosolic DA and DOPA is oxidized via iron catalysis to quinones and semiquinones in the cytosol. 8) Quinones react with cysteine, proteins, and lipids. 9) DA-derived quinones and DA adducts in the cytosol and organelles are phagocytosed in bilamellar autophagic vacuoles/lysosomes where they are permanently stored as NM. 10) Damage from quinone-derived adducts that are not accumulated in NM granules promote neurodegeneration.