Neuromelanin activates microglia and induces degeneration of dopaminergic neurons: implications for progression of Parkinson's disease

Abstract

In Parkinson's disease (PD), there is a progressive loss of neuromelanin (NM)-containing dopamine neurons in substantia nigra (SN) which is associated with microgliosis and presence of extracellular NM. Herein, we have investigated the interplay between microglia and human NM on the degeneration of SN dopaminergic neurons. Although NM particles are phagocytized and degraded by microglia within minutes in vitro, extracellular NM particles induce microglial activation and ensuing production of superoxide, nitric oxide, hydrogen peroxide (H₂O₂), and pro-inflammatory factors. Furthermore, NM produces, in a microglia-depended manner, neurodegeneration in primary ventral midbrain cultures. Neurodegeneration was effectively attenuated with microglia derived from mice deficient in macrophage antigen complex-1, a microglial integrin receptor involved in the initiation of phagocytosis. Neuronal loss was also attenuated with microglia derived from mice deficient in phagocytic oxidase, a subunit of NADPH oxidase, that is responsible for superoxide and H₂O₂ production, or apocynin, an NADPH oxidase inhibitor. In vivo, NM injected into rat SN produces microgliosis and a loss of tyrosine hydroxylase neurons. Thus, these results show that extracellular NM can activate microglia, which in turn may induce dopaminergic neurodegeneration in PD. Our study may have far-reaching implications, both pathogenic and therapeutic.

Fig. 1. Microglia activated by NM produce iROS

(a) NM enhances microglial superoxide production, as measured by electron spin resonance (mean ± s.e.m.; n = 3; * p < 0.05). (b) Measurement of H₂O₂ production from microglial cultures over time indicate that NM (5 μg/ml) elicits H₂O₂ production. This is effectively blocked by the PHOX inhibitor, apocyanin (100 μM). H₂O₂ synthesis is triggered by PMA (100 μM), a known PHOX activator (mean ± s.e.m.; n = 3). (c, d) DCF fluorescence in response to NM, as an indicator of iROS production. (c) PHOX^−/− mice produce less iROS in response to NM (mean ± s.e.m.; n = 8; * p < 0.05; ** p < 0.01). (d) Mac-1^−/− microglia produce less iROS in response to NM (mean ± s.e.m.; n = 6; * p < 0.05).

Fig. 2. Microglia phagocytose and degrade NM

(a) Series of differential contrast images of microglial phagocytosis and degradation of a large particle (~ 14 nm diameter) of NM in ventral midbrain / astrocyte / microglial co-culture at 2 h intervals excerpted from a video http://www.sulzerlab.org/videos/NMmicrogliaDIC.mov. Image acquisition commenced 15 min after addition of NM for 10 h. A microglial cell to the right of the particle at 0 h is in contact with the particle at 2 h. At 4 h, the pigment appears to be absent or nearly so and additional microglia have arrived at the scene. Scale bar = 20 μm. For an example of uptake of NM by microglial filipodia and subsequent NM degradation, see http://www.sulzerlab.org/videos/NMfilopodia.mov. (b, c) The co-cultures prepared as above were fixed 72 h after addition of NM and immunolabeled for dopaminergic neurons by TH (green) and activated microglia by OX-42 (red). Examples of NM particles that have been phagocytosed are indicated by the single-headed arrows. The double-headed arrow indicates an example of a swollen neurite varicosity, which is several-fold larger than typical varicosities in these neurons (average ~1.2 μm, Pothos et al. 1998) which can also be observed. The swollen varicosities are an indicator of toxicity (Cubells et al. 1994). Scale bar = 20 μm.

Fig. 3. Neuronal death induced by NM-activated microglia

(a) NM is toxic to dopaminergic neurons in primary mesencephalic mixed neuron-glia cultures. Rat primary mixed mesencephalic neuron-glia cultures were seeded in 24-well plates at 5×10⁵ /well and treated with 1 – 5 μg NM/ml for 10 days. Effects of NM on the dopaminergic neurons in mixed neuron-glia cultures were assessed by counting the number of dopaminergic neurons after TH staining. Results are expressed as a percentage of the vehicle-treated control cultures (mean ± s.e.m.; n = 3; * p < 0.001). (b) Microglia derived from Mac-1^−/− mice exhibit less NM neurotoxicity (mean ± s.e.m.; n = 4; * p < 0.001). NM toxicity in co-cultures is inhibited in microglia derived from PHOX^−/− mice (c; mean ± s.e.m.; n = 4; * p < 0.001) and by the PHOX inhibitor, apocyanin (100 μM) (d; NM 5 μg/ml; mean ± s.e.m.; n = 7; * p < 0.01).

Fig. 4. Immunostaining in the rat SN 10 days after NM injection

Rats received either phosphate buffered saline or NM at 1 μl/min for 4 min, then the total amount of NM injected into the rat SN was 3.4 μg. The needle was left in place an additional 2 min. Arrows indicate the site of injection within the SN. Representative TH immunostaining of the SN in a NM-injected rat (a), and in a vehicle-injected rat (b). Representative SN Iba-1stained microglia (c, d), GFAP stained astrocytes (e, f), and GABA neurons (g, h) from a NM-injected rat (c, e, g) and from a vehicle-injected rat (d, f, h) are also shown. Scale bar: (a – f) = 440 μm; (g, h) = 175 μm.