This study provides significant support to the hypothesis that NM released from dying neurons can activate microglia, thereby promoting the degeneration of neighboring neurons (Zecca et al. 2003
). Importantly, and in contrast with prior PD models, this study examined the effects of adding NM to cell cultures containing microglia and neurons and injecting NM into rat SN rather than treating cells or animals with exogenous toxins, such as 1-methyl-4-phenylpyridinium ion, 6-hydroxydopamine, l-buthionine-[S, R]-sulfoximine, rotenone or bleomicyn, that are used in other PD models (for review see Dauer and Przedborski 2003
; Hattori and Sato 2007
; Shimohama et al. 2003
). Our cellular system moreover reproduces several morphological and biochemical characteristics present in SN of PD patients. The in vivo
experiment here reported reproduces the cellular conditions occurring in SN of PD patients with extracellular NM, activated microglia and neuronal loss. Both in vitro
and in vivo
systems may thus provide reliable models for studying potential interventions for the chronic phase of PD neuronal death.
NM is a highly insoluble and degradation resistant compound composed of oxidized catecholamines, lipids, peptides and metals that are resident within autophagic / lysosomal organelles of catecholaminergic neurons. We have suggested that NM synthesis is neuroprotective since this process removes excess cytosolic catechols and its derivatives and chelates toxic metals (Sulzer et al. 2000
; Zecca et al. 1994
). NM accumulates over a lifetime in normal individuals because neuronal lysosomes lack the ability to break it down efficiently, a mechanism that may be further compromised during aging (Sulzer et al. 2008
). PD patients, however, loose NM pigment in the SN and locus coeruleus during the course of their disease, an observation that is explained by the microglial degradation pathway introduced here. It is interesting that in our cultures, younger microglia (10 – 20 days after plating) degrade NM within 30 min of phagocytosis while in older microglia (4 weeks) the ability to phagocytose NM is preserved but the degradation of NM is reduced. The decreased capacity to degrade NM could be due to lower production of H2
consequent to a reduced PHOX activity. The behavior of aged microglia may be more similar to that of microglia in SN of PD patients where non-degraded NM granules are present in microglial cell bodies (Banati et al. 1998
; Langston et al. 1999
; McGeer et al. 1988
). An additional reason for a reduced microglial degradation of NM in PD may be that NM organelles remaining after SN cell death in PD are associated with additional components not present in the purified NM used here. Moreover, microglia derived from healthy young rats may be more reactive than those resident in older and diseased subjects.
In addition to the previously reported production of NO, TNF-α, and IL-6, and our present findings of enhanced prostaglandin E2 (data not shown) and MIP-1α, we showed that NM induced production of superoxide, a precursor of reactive species like H2
and indirectly hypochlorite and hydroxyl peroxide. The production of superoxide is dependent on the translocation of cytosolic subunits to the plasma membrane. We observed that NM induced expression of gp91 and H2
, and iROS by microglia in a dose-dependent manner. As these processes were effectively blocked by PHOX inhibitors and much reduced in PHOX−/−
microglia that lack the gp91 subunit and Mac 1−/−
microglia, the data confirm a key role of this Mac-1/PHOX activity in the formation of NM-triggered microglial iROS. The release of inflammatory molecules by NM activated microglia would be expected to contribute to neuronal degeneration by synergizing with cytotoxic agents like H2
, peroxynitrite and proinflammatory cytokines. These inflammatory molecules can diffuse through brain tissue to cause vascular reaction with efflux of blood leukocytes including macrophages into the brain parenchyma, further exacerbating and extending inflammation around blood vessels. Notably, neuropathological studies have demonstrated the presence of vascular damage in SN of PD (Farkas et al. 2000
). Thus, it is likely that microglia associated neurodegeneration produces molecules which can induce vascular release of inflammatory molecules and leukocytes which in turn can produce neuronal damage. This results in a self propelling cycle of neuroinflammation and neurodegeneration.
In our cellular model we introduced a large amount of NM in order to produce rapid and strong microglial activation and extensive neuronal death in 10 days, while in PD the NM is slowly released by dying neurons over years. Although we used the same components present in the SN of PD patients, we reproduced in 10 days in culture a neuronal loss of about 40 % that requires years in human brain. It is remarkable that this acceleration requires only a level of extraneuronal NM in cultures slightly higher than that present in human SN. This concept is further supported by the fact that increasing the amount of NM in the microglia-neuron cultures yields an increased release of ROS, NO, and inflammatory factors along with increased damage to neurons. In our in vivo experiments the injection of human NM into rat SN produced after 10 d an intense microgliosis and loss of a high number of TH neurons.
The amount of NM injected (3.4 μg) was selected based on results from previous study, where we found an extensive microglial activation and neuronal death in rat SN injected with such an amount of NM (Zecca et al. 2008
). This amount of NM was calculated to generate a tissue concentration of NM, slightly higher than that we observed in human SN (Zecca et al. 2002
) since it allows to induce a significant microglial activation and neurodegeneration within 1 – 2 weeks. In our previous study (Zecca et al. 2008
) we used as control an injection of gold microparticles suspension to produce a mechanical effect similar to that of NM particles but without the chemical components of NM. Than the results observed in our studies are specifically due to the chemical components of NM.
The astrocytosis observed in these conditions is a typical response to neuronal damage. It is noteworthy that GABA neurons were not affected either directly by the injected NM or indirectly via microglia activation. This sparing of GABA neurons in presence of an extensive loss of TH neurons makes our in vivo system a promising candidate model of PD. In our in vivo context, a localized high concentration of extracellular NM was produced with injection of human NM into rat SN. With this procedure we obtain in ten days a localized neuronal loss whose degree is similar to that taking place during years in the entire SN of PD subjects. Obviously further experiments will be necessary for a full validation of this concept / setup as an animal model of PD.
The present data demonstrate that extracellular NM in the absence of microglia is not itself toxic for neurons. However, once released from dying neurons, NM activates microglia, in part due to recognition by the Mac-1 integrin receptor to further promote neurotoxity. As the treatment with apocyanin that inhibited PHOX-dependent iROS also reduced neurotoxicity, a portion of the toxicity due to NM microglial activation is due to the activity of this enzyme. Significant work will be required to determine which of the many other potential additional pathways activated by NM in microglia cause neurotoxicity. Notably, the activated microglia appear to make extensive contact with neurons, and it may be that the toxicity is highly localized, perhaps by release of toxic compounds at zones of tight adhesion. If so, this process, which is not in itself selective for catecholamine neurons, may induce chronic neuronal death particular to the SN and locus coeruleus, since these catecholaminergic nuclei, which are severely damaged in PD, are also those with the highest NM content in the brain. There are several factors contributing to the high vulnerability of DA neurons of SN in PD. It is worth noting that SN has the highest density of microglia in the brain (Kim et al. 2000
). Moreover in DA neurons of SN there is a high concentration of DA which can be oxidized to generate reactive/toxic quinones. The low glutathione content corresponding to low antioxidant ability and the high load of organic toxins and toxic metals into NM further make DA neurons of SN particularly vulnerable (Zecca et al. 2004
Considering that a continuous and high release of NM by dying neurons of SN occurs in PD, and that NM is insoluble and stable for long periods in the extracellular space these results suggest that extraneuronal NM can induce a sustained microglia activation and ensuing neurodegeneration in PD.