When dopaminergic neurons are damaged, chronic microglial activation and the persistent selective loss of dopaminergic neurons can occur, long after the initial toxic insult has abated. This persistent cellular response called reactive microgliosis is widely believed to be a predominant mechanism underlying progressive neuron damage in neurodegenerative diseases. Here we specifically addressed why reactive microgliosis in response to dopaminergic neuron damage is chronic and toxic.
We used an in vitro
model to separate the soluble signals released by damaged dopaminergic neurons from the toxicant causing the damage (MPP+
). This approach allowed analysis of the dopaminergic neuron survival in response to only the neuron injury signals, revealing that dopaminergic neurons are inherently more susceptible to soluble neuron injury signals when compared to other neuronal-sub-types and that microglia and NADPH oxidase are key to the mechanism of damage. These data strongly support that dopaminergic neurons may be inherently susceptible to reactive microgliosis and relentless propagation of the chronic and neurotoxic response continuing on in the absence of the initial toxic stimulus. Given that the substantia nigra contains both the population of dopaminergic neurons selectively lost in Parkinson’s disease and a disproportionately high concentration of microglia (4.5 times as many as other regions of the brain) (Kim et al.
), our findings indicate that damaged neurons themselves are culpable in propagating further neurotoxicity with pro-inflammatory signals to microglia (), providing a key insight into the chronic and selective nature of Parkinson’s disease.
Figure 7 μ-Calpain is a key factor driving the progressive nature of dopaminergic neuron damage. Dopaminergic (DA) neuron damage is chronic in part because damaged cells release soluble factors that accumulate over time to active resident microglia, driving (more ...)
After isolating the soluble-neuron injury factors released by damaged dopaminergic neurons and confirming that they were selectively toxic through microglial activation and the super-oxide producing enzyme, NADPH oxidase, we then began to identify which soluble neuron injury factors were responsible for microglial activation and consequent dopaminergic neuron damage. In the present study, we chose a hypothesis-directed approach and investigated whether μ-calpain is a key neuron injury signal causing reactive microgliosis and consequent chronic dopaminergic neuron damage.
Calpain is a family of calcium-dependent cysteine proteases that have been implicated in chronic cellular damage, e.g. extracellular calpain in the liver (Limaye et al.
; Mehendale and Limaye, 2005
). While calpain is traditionally viewed as an intracellular protease, it is found extracellularly in human disease and tissue damage, such as arthritis (Fushimi et al.
) and liver damage (Limaye et al.
; Mehendale and Limaye, 2005
). In fact, extracellular calpain is present in the supernatant from damaged cortical neurons in vitro
(Siman et al.
), supporting a potential role for this protease as a neuron injury signal. Notably, while the mechanisms remain unclear, calpain has been linked to intracellular process of dopaminergic neuron death (apoptosis and necrosis) (Crocker et al.
), microglial activation (Shields et al.
), and Parkinson’s disease (Mouatt-Prigent et al.
; Crocker et al.
). Intracellularly, calpain activity is shown to increase in dopaminergic neurons damage by MPP+
and MPTP in vitro
and in vivo
, where calpain inhibitors can attenuate MPP+
-induced dopamine cell loss (Crocker et al.
). Calpain is also upregulated in post-mortem Parkinson’s disease brain, verifying that calpain is present in Parkinson’s disease brain and is upregulated upon dopaminergic neuron damage (Crocker et al.
). However, until the present study, the role of extracellular calpain in dopaminergic neuron damage and its effect on chronic and toxic microglia activation were unknown.
Calpain has multiple isoforms, with μ- and m-calpain ubiquitously distributed in the cytoplasm of all cells. Intracellular calpain is involved in numerous cellular functions, including membrane trafficking, receptor signalling, inflammatory signalling, apoptosis and necrosis (Franco and Huttenlocher, 2005
). Calpain’s intracellular functions are critical for both survival and death, making the tight regulation of calpain activity essential. The dysregulation of calpain has been associated with numerous diseases, such as arthritis (Fushimi et al.
), Alzheimer’s disease (Zatz and Starling, 2005
), multiple sclerosis (Sloane et al.
; Guyton et al.
), optic neuritis (Shields and Banik, 1998
) and Parkinson’s disease (Crocker et al.
While most studies have focused on the intracellular role of calpain in cell death, extracellular calpain has also been documented (Nishihara et al.
; Xu and Deng, 2004
) and implicated in disease. For example, calpain is found in the synovial fluid of arthritis patients (Fushimi et al.
) and has been associated with degeneration of the myelin sheath (Shields et al.
; Sloane et al.
). Calpain is reported to be released extracellularly in the case of tissue damage in liver (Limaye et al.
; Mehendale and Limaye, 2005
) and cortical neuron damage (Siman et al.
). Interestingly, calpain and the breakdown products of its substrates have been found in the cerebral spinal fluid after acute ischaemia and are proposed to be markers for neuronal damage (Siman et al.
). Once in the extracellular space, calpain is presumed to be highly active and unregulated, as extracellular calcium concentrations (1.3 mM) (Limaye et al.
) are higher than the μM (μ-calpain) and the mM (m-calpain) required for activation. In the case of liver injury, once outside of the cell, calpain is believed to attack the membrane of surrounding cells to result in propagation of tissue damage (Limaye et al.
; Mehendale and Limaye, 2005
). Alternatively, several extracellular proteases are reported to be pro-inflammatory when extracellular, which may result in neuronal death (Choi et al.
; Kim et al.
), but the mechanisms remain poorly described.
In this study we show that μ-calpain is a key soluble neuron injury factor driving reactive microgliosis. μ-Calpain is present in the conditioned medium from MPP+
-damaged dopaminergic neurons (N27 cells), where this conditioned medium was shown to be selectively toxic to dopaminergic neurons through microglial NADPH oxidase. Extracellular μ-calpain added to cultures activated microglia, as evidenced by changes in morphology and superoxide production. Furthermore, μ-calpain-induced neurotoxicity was selective for dopaminergic neurons and occurred only in the presence of microglia. Finally, μ-calpain only exerted dopaminergic neurotoxicity in cultures with functioning NADPH oxidase, indicating that superoxide production was the predominant mechanism of μ-calpain-induced neuron damage. Thus, we show that μ-calpain is a soluble neuron injury factor that is selectively toxic to dopaminergic neurons through microglia-generated oxidative insult, revealing key mechanisms of chronic neurodegenerative pathology (), as reactive microgliosis may contribute to neuronal damage in diverse neuronal diseases (Block and Hong, 2005
, 2007; Block et al.
While μ-calpain may be a key signal that damaged dopaminergic neurons release to activate microglia and propagate damage, reactive microgliosis is a complex phenomenon and there are probably multiple factors released that contribute to the toxic microglial response. In fact, previous studies have reported other damage signals implicated in toxic reactive microgliosis, such as α-synuclein and neuromelanin (Block and Hong, 2007
). However, μ-calpain is ubiquitously expressed in all cell types and previous reports indicate that it is also released from damaged cortical neurons (Siman et al.
), suggesting that μ-calpain may be a general factor of reactive microgliosis. The dopamine selective toxicity of μ-calpain and the conditioned medium that we report here is likely to be due to the characteristic selective vulnerability of dopaminergic neurons to microglial activation and consequent production of reactive oxygen species, rather than a dopaminergic neuron-specific signal. For example, previous reports have shown that multiple other toxins are selectively toxic to dopaminergic neurons through microglial reactive oxygen species production, such α-synuclein, neuromelanin, lipopolysaccharide, paraquat, air pollution, rotenone and substance P (Block and Hong, 2007
). Our current results indicate that μ-calpain and the soluble neuron-injury signals contained in the conditioned medium from damaged dopaminergic neurons converge on this basic mechanism of selective dopaminergic neurotoxicity. Thus, while reactive microgliosis may underlie diverse neurodegenerative diseases, our study suggests that dopaminergic neurons are more likely to be negatively affected than other cell types, providing much needed insight into the progressive nature of Parkinson’s disease. Indeed, this study supports the premise that dopaminergic neurons may be particularly vulnerable to the chronic effects of a single neurotoxic insult that propagates because of the microglial response to neuronal injury, which then becomes the driving force of persistent and progressive dopaminergic neuron damage.