ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo

Abstract

The role of microglial motility in the context of adult neurodegeneration is poorly understood. In the present work, we investigated the microanatomical details of microglia-neuron interactions in an experimental mouse model of Parkinson's disease following the intraperitoneal injection of MPTP. The specific intoxication of dopaminergic neurons induces the cellular polarization of microglia, leading to the formation of body-to-body neuron-glia contacts, called gliapses, which precede neuron elimination. Inhibiting ROCK/Cdc42-mediated microglial motility in vivo blocks the activating features of microglia, such as increased cell size and number of filopodia and diminishes their phagocyting/secreting domains, as the reduction of the Golgi apparatus and the number of microglia-neuron contacts has shown. High-resolution confocal images and three-dimensional rendering demonstrate that microglia engulf entire neurons at one-to-one ratio, and the microglial cell body participates in the formation of the phagocytic cup, engulfing and eliminating neurons in areas of dopaminergic degeneration in adult mammals.

Figure 1. Microglial cells polarize their processes toward neurons after MPTP.

(A) Confocal images of the SNpc show the activation of microglial cells, characterized through the increase of their cell body size and the higher number of terminal tips in close apposition to dopaminergic neurons after MPTP treatment. (B) The confocal pictures from the SNpc were analyzed in detail and representative illustrations of the cells were delineated based on the confocal images. Scale bar in A and B: 50 μm. (C) The number of terminal tips of microglial cells increased after MPTP treatment. *p<0.05. (D) Microglial cell processes and cell bodies are in contact with dopaminergic neurites and dopaminergic cell bodies. (D1) Confocal image showing microglial cells (green) contacting their processes with dopaminergic neurons (red). Cell nuclei are counterstained with DAPI (blue). Arrows indicates contacts with dopaminergic cell bodies or neurites. Scale bar in 1: 25 μm. (D2) A microglial cell body in contact with a dopaminergic neurite. (D3) A microglial cell body in contact with a dopaminergic neuron cell body. Scale bar in 3: 20 μm. (E) The number of microglial cells processes in contact with dopaminergic processes [Pr-Pr] and with dopaminergic cell bodies [Pr-B] increases after MPTP treatment. The contacts between microglial cell bodies and dopaminergic structures (B-Pr and B-B) were not modified. (TOTAL/Pr = [Pr−Pr]+[B−Pr]), TOTAL/Ne = [Pr−B]+[B−B]. *p<0.05 (F) Quantification of the length of the contact between microglial processes and dopaminergic neurites or dopaminergic cell bodies increases after MPTP treatment. *p<0.05, **p<0.01.

Figure 2. Blockade of ROCK prevents microglial polarization.

(A) HA-1077 inhibits the increase of ROCK activity induced by MPTP. *p<0.05 compared with the control. (B) Top panel shows representative confocal pictures of Iba-1 at the level of the SNpc of the four different groups. Bottom panel shows illustrations of two representative microglial cells from each group obtained from the Iba-1 confocal images. (C) The polarization of microglial cells induced through MPTP treatment is abolished via the specific ROCK inhibitor HA-1077. The increased microglial size and the increased number of microglial processes induced through MPTP treatment are diminished via the specific ROCK inhibitor HA-1077. *p<0.05, **p<0.01 with respect to the control. (D) The increased number of contacts between microglial processes and dopaminergic neurites (Pr-Pr) and cell bodies (Pr-B) induced through MPTP treatment is abolished with specific ROCK inhibitor HA-1077. (E) Western immunoblot of Cdc42 in the nigrostriatal pathway of mice treated with saline, HA-1077, MPTP or MPTP+HA-1077. Human platelet extract (HPE) was used as a control signal for Cdc42. (F) Quantification of the optical density of Cdc42 normalized with GAPDH. Note that HA-1077 inhibits the increase of Cdc42 induced through MPTP treatment, as measured using Western Blot **p<0.01, *p<0.05 compared with the control.

Figure 3. F-actin and Cdc42 is highly clustered in microglial cells.

(A) Stack of confocal sections of the nigrostriatal area immuno-stained for Iba-1 (green), stained with 633-phalloidin (Magenta) to label F-actin, and counterstained with DAPI (blue) in a MPTP-treated mouse. In the Overall view, because F-actin is ubiquitous, high exposure to infrared 633-nm laser displays the positive stain of the tissue but hampers the appreciation of details. However, low exposure to the 633-nm laser reduces the intensity of the general F-actin staining and facilitates the appreciation of high fluorescent clusters. Panel on the center shows the Detail of these stacks of images. F-actin highly fluorescent clusters are observed in microglial cells but not in the rest of neighboring cells. Panel on the right shows the Z-axis view, which facilitates the identification of the specific clustering of F-actin in microglia. (B) Stack of confocal sections of the nigrostriatal area immuno-stained for Iba-1 (green) and Cdc42, and counterstained with DAPI (blue) in an MPTP-treated mouse. In the Overall view because Cdc42 is also ubiquitous, high exposure to the 594-nm laser shows the general immuno-staining of the tissue but impedes the appreciation of the details. However, low exposure to the 594-nm laser reduces the intensity of the general Cdc42 staining and facilitates the appreciation of highly fluorescent clusters. The Detail of these stacks of images in the central panel shows that Cdc42 highly fluorescent clusters are observed in microglial cells and not in the rest of neighboring cells. The Z-axis view on the right panel facilitates the identification of the specific clustering of Cdc42 in microglia. The arrows indicate representative microglial clusters. (C) Confocal analysis of polarization and clustering of F-actin in a microglial cell contacting a dopaminergic neuron after MPTP injection. F-actin cluster appear at the protrusion of polarized microglial cells toward the dopaminergic cell body. (D) Explicative diagram of the image from C.

Figure 4. HA-1077 treatment prevents gliaptic body-to-body contacts and neuron elimination.

(A) Quantification of the number of dopaminergic neurons of the SNpc of mice treated with Saline (Control), MPTP, HA-1077 or MPTP + HA-1077. The treatment with HA-1077 prevents the dopaminergic degeneration induced by MPTP. (B) Representative images of immuno-staining for TH⁺ neurons to observe dopaminergic neurons of the SNpc of a representative mouse of each experimental group analyzed. (C) Illustration of a characteristic gliaptic body-to-body contact between microglia and TH neurons. The augmented detail of the merged image is shown rotated in the vertical picture. (D) Quantification of the number of body-to-body contacts between dopaminergic neurons and microglial cells of the SNpc of mice treated with Saline (Control), MPTP, HA-1077 or MPTP+HA-1077. The formation of body-to-body contacts precedes neuronal degeneration and conversely treatment with HA-1077 prevents the formation of body-to-body contacts after MPTP treatment.

Figure 5. Microanatomy of engulfing gliapses formation in the SNpc of Parkinsonian mice.

(A) Image of a gliapse in the SNpc of an MPTP-treated mouse (Giapse 1). The microglial cell (red) is in close apposition with a dopaminergic neuron (white) and its processes are polarized to the dopaminergic neuron cell body. (B) Three-dimensional rendering of Gliapse 1. (C) Image of gliapse formation in the SNpc of an MPTP-treated mouse. (D) Three dimensional rendering of Gliapse 2. (E) Analysis of polarization of Golgi and Iba-1 at the engulfing gliapse. Optical sections (0.5 μm thick) from Gliapse 2 are shown in the top row. Immunofluorescence for the Golgi apparatus (GM130, green), microglia (red) and TH (white) are displayed, together with nuclear counterstaining using DAPI (blue). GM130 is arranged at the interface. The bottom row shows the details of the interface area. Iba-1 appears polarized toward the dopaminergic neuron (1). Microglial GM130 is polarized only on the side of the cell in contact with the dopaminergic neuron (2, 3). GM130 occupies the area of the intercellular interface (4, 5). Ratio of the physiological activity analysis of the channels shows the maximum activity at the engulfing gliapses interface (6). (F) Confocal analysis of the polarization of Golgi in the xy plane and along the z-axis. Polarization of GM130 can be observed in the lateral views along the z-axis scanning. Scale bar in A; 25 μm, in B; 15 μm.

Figure 6. Microglial cells phagocyte degenerating neurons in the SNpc (A) Confocal images of engulfing events in the SNpc of mice after MPTP-treatment.

Microglial processes engulfing a dopaminergic neuron after MPTP treatment (1, 1′, 2, 2′). Engulfed dopaminergic neurons present a smaller size, low intensity of TH and pycnotic nuclei (2, 2′). Microglial cells engulfing pycnotic nuclei (3, 3′). (B) Diagrams of the three events described in A (1″, 2″ and 3″). (C) The subtraction of the black background in the engulfing gliapse shown in 2 and 2″ facilitates the observation of the pycnotic nature of the nucleus of the neuron, the punctuated TH staining and the shape of the engulfed dopaminergic neuron. (D) Lateral view of the z-axis of an engulfing microglia. Note that the pycnotic extra nucleus is completely surrounded by the Iba-1⁺ membrane. (E) Three-dimensional reconstructions of engulfing microglial cells in the SNpc after MPTP treatment. In both examples, the extra nucleus is completely surrounded by the membrane of the microglial cell. The clipping plane (yellow) is shown at the level of the extra nucleus. (F) Overhead view of a three-dimensional reconstruction of a microglial cell engulfing an extra nucleus in the SNpc of a mouse after MPTP treatment in the SNpc.

Figure 7. Localization of lysosomes in engulfing microglia.

(A) Confocal analysis of an engulfing microglial cell stained with Lectin (red), showing Cathepsin-D⁺ lysosomes (green) in the SNpc of a mouse treated with MPTP. DAPI (blue) was used as a counterstaining. (B) Lateral view of the extra-nucleus inside the microglial phagosome from A. (C) Detail of Cathepsin-D⁺ lysosomes close to the microglial nucleus. The images were treated with a surface filter to better appreciate the cellular structures. (D) Examples of engulfing microglia with Cathepsin-D⁺ lysosomes. An image filter was applied to highlight the contours in the three examples. (E) Detailed confocal analysis of an engulfing microglia showing a kidney-shaped nucleus surrounding the Cathepsin-D lysosome. The pycnotic engulfed extra nucleus can also be clearly observed with the DAPI counterstaining.

Figure 8. Actin clustering in engulfing microglia.

(A) F-Actin accumulation can be observed in the vicinity of microglial nucleus in engulfing gliapses in the SNpc of Parkisonian mice. Two examples of gliapses are shown, where a microglial cell engulfs an extra nucleus and shows F-actin accumulation (arrows). Higher magnifications of the F-actin accumulation are shown in 1, 1′, 2 and 2′. Explicative diagrams are shown on the right. (B) Example of F-actin accumulation in other engulfing gliapse. Higher magnifications of the F-actin accumulation are shown in 3 and 3′. In this case, the extra nucleus is located at a different optical plane and the lateral view along the z-axis is shown in D. In all three gliapses actin clusters are moved away from the interface phagosome-microglial nucleus. (C) The 3D analysis of the level of relative fluorescence of F-actin in the three gliapses studied. The maximum level of F-actin fluorescence was detected specifically in the engulfing gliapses (magenta arrows). (D) Lateral z-axis view and three-dimensional reconstruction of Gliapse 3. Overview of the 3D reconstruction is shown in 1. The clipping plane at the level of F-actin accumulation of the 3D reconstruction is shown in 2. The yellow insert in 2 is magnified in 3.