| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 6, 2004 as doi:10.1096/fj.03-0374fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Neuroscience Research Center, Medical Faculty (Charité), Humboldt University, Berlin, Germany
2 Correspondence: Research Institute of Environmental Medicine, Heinrich Heine University, Auf’m Hennekamp, 50 40225 Düsseldorf, Germany. E-mail: tilman.grune{at}uni-duesseldorf.de
SPECIFIC AIMS
Neuronal loss via apoptotsis is a key element in numerous neurodegenerative diseases. It has been suggested that microglial cells are phagocytosing and degrading apoptotic material. In this study we investigated the ability of microglial cells to take up and degrade neuronal apoptotic material, demonstrated a CD36 receptor mediated pathway of the uptake, and a lysosome and proteasomal mediated degradation of proteins from apoptotic material.
PRINCIPIAL FINDINGS
1. Proteins from apoptotic material are degraded within microglial cells
To investigate whether apoptotic bodies from neuronal cells are phagocytosed by microglia, we used a common apoptosis model of PC12 cells. These cells were brought into an apoptotic state by growth factor depletion. To investigate the fate of proteins originated from PC12 cells, we grew them in a medium containing [35S]-methionine, induced apoptosis, and isolated apoptotic bodies. Apoptotic bodies were added to cultures of rat primary microglial cells and liberation of free amino acids was followed over time. Cultures of microglial cells were able to degrade the proteins originated from apoptotic bodies (Fig. 1
A). Within 24 h almost 30% of added radiolabeled proteins were degraded. To check whether this effect was due to microglial cells and not to remaining proteolytic activity within the apoptotic bodies originating from the PC12 cells, we tested release of acid soluble radioactivity from the protein pool of apoptotic bodies in tissue culture medium without presence of microglial cells. There was no substantial degradation without microglial cells (circles, Fig. 1A
). Additionally, we found that nonapoptotic PC 12 cells were only degraded to a minor extent.
|
Since a wide variety of cytokines can activate microglia and this activation process may influence the uptake and degrading capacity of microglial cells, we used primary microglial cells activated by various cytokines or components and measured their ability to degrade proteins from apoptotic bodies. All microglia activation components used were able to stimulate the ability of microglial cells to degrade apoptotic body borne proteins (Fig. 1B
). LPS and PMA were able to nearly double the degradation rate. We used tissue culture medium preconditioned by nonactivated or activated microglial cells and tested its protein degrading capacity to determine whether enhanced protein degradation takes place intracellularly or is due to release of various proteolytic enzymes into the extracellular environment. A minimal amount of proteolysis of proteins originating from apoptotic bodies was due to extracellular components (Fig. 1C
). This was also true of medium preconditioned with activated microglial cells, although LPS and PMA showed some increase of cell culture medium proteolytic activity.
2. The degradation of apoptotic bodies can be inhibited by competitive material
Next, we tested whether degradation of proteins from apoptotic material can be inhibited by other vesicles or oxidized proteins. We were able to demonstrate that vesicles produced from nonapoptotic PC12 cells, oxidized LDL, and oxidized myelin basic protein are able to reduce the degradation of proteins from apoptotic material. This suggests a common rate-limiting step in uptake and degradation of apoptotic material with other extracellular material. Therefore, we tested the receptors involved in uptake of apoptotic material and proteolytic systems involved in degradation of proteins taken up by microglial cells. Because a central role of the CD36 scavenger receptor has been proposed, we postulated a function of this receptor in uptake of apoptotic material.
3. The CD36 receptor plays a key role in the uptake of apoptotic material by microglial cells
To determine the role of the CD36 receptor in uptake of apoptotic material, we first tested the effect of receptor blocking antibodies on the degradation of proteins from apoptotic material. It was demonstrated that using two different antibodies against the CD36 receptor was able to block uptake of apoptotic material by more than 50%. To obtain further information about the role of the CD36 receptor, we isolated microglial cells from a CD36 mutant rat strain (mtCD36). In this mutant the CD36 receptor is present but not functional. In microglial cells isolated from the mtCD36 rat strain, degradation of proteins from apoptotic material was also reduced by 
50%. Using fluorescence labeled apoptotic bodies from PC12 cells, we demonstrated a clear reduction of uptake of apoptotic material in mtCD36 derived microglial cells in comparison to normal microglia. Activation of mtCD36 microglia did not increase degradation of proteins from apoptotic material in these cells, indicating uptake as an important (and perhaps rate-limiting) step in removal of apoptotic material from the extracellular environment.
4. Proteins of apoptotic bodies are degraded by both lysosomal proteases and the proteasomal system
Inhibitor experiments revealed that lysosomal proteases are involved in degradation of proteins derived from apoptotic bodies. This clearly suggests transport of some predegraded material from the vesicular/lysosomal compartment into the cytosol. Use of both inhibitors was able to block the degradation of proteins from apoptotic bodies by 75%, indicating the key role of these two proteolytic systems in degradation of apoptotic material. Inhibition of the proteasomal system is accompanied by some accumulation of proteins from apoptotic bodies within microglial cells.
5. Blockage of further receptors
To determine which other receptors are involved in uptake and degradation of apoptotic vesicles, we tested lectin, vitronectin, and phosphatidylserine. Blockage of these receptors only reduced degradation of apoptotic vesicles in nonstimulated cells to a minor extent. Degradation blockage of apoptotic vesicles was significant for all tested receptors in stimulated cells. Blockage of all these receptors (including CD36) was able to inhibit uptake of apoptotic material by 75% or more.
6. Activation of microglial cells has no influence on proteolytic activities and CD36 expression
To ascertain whether activation of microglial cells and enhanced degradation of proteins is derived from apoptotic bodies, we tested whether either the proteolytic systems or the CD36 receptor are induced. We could show no induction of lysosomal proteolytic capacity occuring due to activation of microglial cells in our system. On the other hand, several inducers of microglial activation, including 
-interferon, were able to increase proteasomal activity. No correlation could be established between proteasomal activation and enhanced degradation of proteins from apoptotic bodies by microglial cells. Next, we tested the expression of the CD36 receptor. Only a limited induction of the CD36 receptor could be demonstrated, suggesting the role of additional uptake mechanisms in activated microglia.
CONCLUSIONS AND SIGNIFICANCE
Microglial cells, as residential macrophages of the brain, seem to be able to recognize a wide range of extracellular material, take this material up, and degrade it. We were able to demonstrate that this also includes apoptotic bodies of neuron-like cells.
We demonstrated the ability of microglial cells to take up and degrade apoptotic material. It could be shown clearly that major degradation takes place inside the microglial cell and that extracellular degradation pathways are quantitatively less important. Uptake is mediated by 50% by the CD36 scavenger receptor. Uptaken material is degraded within microglial cells and proteins from apoptotic bodies are degraded from lysosomal proteases as well. In an activated state microglial cells are able to increase their uptake and degradation of apoptotic material. Due to their ability to increase the digestion of apoptotic material after activation, microglial cells are uniquely adapted to this clearance function.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0374fje; doi: 10.1096/fj.03-0374fje 
This article has been cited by other articles:
|
|
 |
|
  J. Neumann, S. Sauerzweig, R. Ronicke, F. Gunzer, K. Dinkel, O. Ullrich, M. Gunzer, and K. G. Reymann Microglia Cells Protect Neurons by Direct Engulfment of Invading Neutrophil Granulocytes: A New Mechanism of CNS Immune Privilege J. Neurosci., June 4, 2008; 28(23): 5965 - 5975. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. S. Kim, S. S. Kim, J. J. Cho, D. H. Choi, O. Hwang, D. H. Shin, H. S. Chun, M. F. Beal, and T. H. Joh Matrix Metalloproteinase-3: A Novel Signaling Proteinase from Apoptotic Neuronal Cells That Activates Microglia J. Neurosci., April 6, 2005; 25(14): 3701 - 3711. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
(200 U/mL), PMA (1 µM) and LPS (10 µg/mL). All stimulations were performed for 16 h. Data represent mean ± SD of three experiments. C) Proteolysis of apoptotic bodies in preconditioned medium derived from 16 h activated primary microglial cells. Medium was removed from activated cells, centrifuged and added to apoptotic bodies under conditions described above. Data represent mean ± SD of three experiments.