Overexpression of the immunoreceptor CD300f has a neuroprotective role in a model of acute brain injury

Abstract

It is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). We have analyzed the function of cluster of differentiation (CD)300f immunoreceptor in a model of excitotoxic rat brain damage. First, to explore the presence of endogenous ligand(s) for this receptor we used a human CD300f-Ig soluble protein and confocal microscopy, showing specific staining mainly in CNS white matter and on the surface of oligodendrocytes and certain astrocytes. Next, we demonstrated in a model of in vivo rat brain excitotoxic damage that the overexpression of human CD300f induced a significant reduction in the lesion volume. To validate these results, we cloned the rat ortholog of CD300f protein (rCD300f). The overexpression of rCD300f receptor had a comparable neuroprotective effect after the acute brain injury and a similar CNS staining pattern when stained with the rCD300f-Ig soluble protein. Interestingly, when we analyzed the expression pattern of rCD300f in brain cells by quantitative polymerase chain reaction and immunohistochemistry, we detected the expression of CD300f as expected in microglial cells, but also in oligodendrocytes and neurons. These data suggest that the neuroprotective role of CD300f would be the result of a complex network of cell interactions.

Figure 1

Localization of the putative ligand(s) of hCD300f in vitro and in vivo. Primary cultures of the different glial central nervous system (CNS) cell types (A–E) or sections from mouse and rat brain (mouse brain, F–I) were used for co‐localization studies of hCD300f‐IgG2a (10 µg/mL) fusion protein and different cell‐specific markers. Small undifferentiated oligodendrocytes with few ramifications (A, GalC) and large highly ramified differentiated oligodendrocytes (B, GalC; C, myelin basic protein [MBP]) showed specific punctate staining with the fusion protein. Glial fibrillary acidic protein‐(GFAP) positive polygonal protoplasmic astrocytes did not show specific staining with the fusion proteins (D, arrow), being only positive the ramified fibrous GFAP‐stained cells. Microglial cells were negative for the staining with the fusion protein (E). A similar punctuate staining pattern was observed by confocal microscopy in the white matter of the brain stained with the fusion protein, which did not co‐localized with myelin markers (stained with CNPase, F, corpus callosum) but co‐localized with the oligodendrocyte soma and proximal projections (stained with APC, arrows in G–I). Scale bars: F–I 10 µm.

Figure 2

Increased staining for the ligand(s) of CD300f in lesioned rat brain. Normal brain (A,B) showed a punctate staining with hCD300f‐IgG2a fusion protein mainly in white matter areas like corpus callosum, external capsule (B: arrow) or the anterior commisure (B: arrowhead). Brains subjected to intra‐striatal injection of N‐methyl‐D‐aspartate (NMDA) showed an increased hCD300f‐IgG2a staining in the lesion core (C,D) in comparison with the noninjured brain (A,B). Representative higher magnification images from the sections in B and D (open squares) are shown in A and C. No staining was observed when sections were incubated with control mouse IgG2a. Scale bar: 10 µm.

Figure 3

Overexpression of hCD300f is neuroprotective after and acute brain injury. Striatal injection of N‐methyl‐D‐aspartate (NMDA) induced a well‐delimitated lesion in the striatum and the cortex when observed by Nissl staining 3 days post‐intervention (A,B). At this time point, the measurement of the % of lesioned hemisphere (C, black lines in A,B) showed that the overexpression of hCD300f using the NLSCt vector induced a significant neuroprotection (P < 0.05) when compared with the overexpression of a control transgene (EGFP).

Figure 4

Cloning of Rattus Norvegicus CD300f. A. Nucleotide and predicted amino acid sequences of rCD300f (GU057984). The predicted amino acid sequence is shown below the nucleotide sequence. The putative signal peptide is double underlined, the Ig‐like domain is in bold type, the transmembrane domain is underlined (dotted line), and the consensus immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐like sequences are underlined (single line). The N‐glycosylation site is boxed, and cysteine residues involved in the Ig‐like fold are circled. B. Protein sequence alignment of human and rat CD300f. Identical amino acids are shown on black background and similar residues are on gray background.

Figure 5

Expression of rCD300f in CNS primary cultures. Real–time polymerase chain reaction (PCR) (A) fluorescence‐activated cell sorting (FACS) (B) and immunocytochemistry (C–H) of different central nervous system (CNS) primary cultures were performed to analyze the expression of rCD300f. Microglial cells (Mic), undifferentiated oligodendrocytes (Olig) and hippocampal neurons (Neu) expressed mRNA for rCD300f while astrocytes (Ast) did not. Treatment of microglia with lipopolysaccharide (LPS) (1 µg/mL) plus IL1β (10 ng/mL) induced an increase in the mRNA for rCD300f (A, P < 0.05). Its expression was also detected in parental and rCD300f transfected RBL‐2H3 cells by FACS staining, whereas staining with an anti‐CLM7 antibody was negative. Isotypic antibody (grey histograms), anti‐CLM1/CLM7 (white histograms) (B). Microglia (stained with C: Iba1 or D: CD11b), differentiated oligodendrocytes (stained with E: GalC or F: myelin basic protein [MBP]) and hippocampal neurons (G) were positive by immunocytochemistry. Astrocytes were negative (big ovoid nuclei, arrows in B). No staining was observed in the absence of primary antibody (H, control IgG2a). Scale bars: 20 µm.

Figure 6

Localization of the putative ligand(s) of rCD300f in vitro and in vivo. Sections from mouse and rat brain were incubated with the fusion proteins rCD300f‐IgG2a for the localization of the putative ligand(s) of CD300f, and confocal co‐localization studies were performed with cell‐specific markers (C–H) or with the Thy1‐YFP‐H mice (I–K). Staining with the fusion proteins was observed mainly in the brain white matter as in the external capsule (A, external capsule (EC); Cx = neocortex; St = striatum; V = ventricle) or the fiber tracts of the striatum (B, St.). The punctuate staining pattern co‐localized with the oligodendrocyte soma and proximal projections (stained with APC, arrows in C–E, corpus callosum) but not with myelin itself (stained with CNPase, F, striatum). No colocalization could be observed with microglia or blood vessels (stained with TL, G). Cortical neuron cultures were positive for rCD300f‐IgG2a staining (stained with β3‐tubulin, H). For extensive in vivo neuronal labeling, Thy1‐YFP mice were used, and no co‐localization was detected between YFP neuronal soma, dendrites, spines or axons and the fusion proteins (I: striatum; J: neocortex, red staining is nonspecific capillaries staining; K: corpus callosum). Scale bars: A,B: 50 µm, C–K: 10 µm.

Figure 7

Localization of the putative ligand(s) of rCD300f in spinal cord in vivo. Spinal cord sections from Thy1‐YFP mice were stained with rCD300f‐IgG2a fusion protein, and confocal microscopy was used to visualize specific staining. Corticospinal tract axons can be visualized in green. The red staining for rCD300f‐IgG2a showed a punctate pattern similar to that observed in the brain, and was also mainly present in the white matter tracts of the spinal cord. G–I are higher magnification images from the dorsal corticospinal tract (square area shown in E). No co‐localization was observed between green and red staining. VGM = ventral grey matter; DGM = dorsal grey matter.

Figure 8

Overexpression of rCD300f is neuroprotective after and acute brain injury. Striatal injection of N‐methyl‐D‐aspartate (NMDA) induced a well‐delimitated lesion when observed by Nissl staining 3 days post‐intervention. At this time point, the measurement of the % of lesioned hemisphere showed that the overexpression of rCD300f using the NLSCt vector induced a significant neuroprotection (P < 0.05) when compared with the overexpression of a control transgene (EGFP).