Spatiotemporal Expression of GRP78 in the Blood Vessels of Rats Treated With 3-Nitropropionic Acid Correlates With Blood–Brain Barrier Disruption

Abstract

Glucose-regulated protein (GRP78) or BiP, a 78-kDa chaperone protein located in the endoplasmic reticulum (ER), has recently been reported to be involved in the neuroglial response to ischemia-induced ER stress. The present study was designed to study the expression patterns of this protein and the cell types involved in the induction of GRP78 expression in rats treated with the mitochondrial toxin 3-nitropropionic acid (3-NP). GRP78 immunoreactivity was almost exclusively localized to striatal neurons in saline-treated controls, but GRP78 expression was induced in activated glial cells, including reactive astrocytes and activated microglia/macrophages, in the striata of rats treated with 3-NP. In the lesion core, increased GRP78 immunoreactivity was observed in the vasculature; this was evident in the lesion periphery of the core at 3 days after lesion induction, and was evenly distributed throughout the lesion core by 7 days after lesion induction. Vascular GRP78 expression was correlated, both temporally and spatially, with infiltration of activated microglia into the lesion core. In addition, this was coincident with the time and pattern of blood–brain barrier (BBB) leakage, detected by the extravasation of fluorescein isothiocyanate-albumin, an established BBB permeability marker. Vascular GRP78-positive cells in the lesion core were identified as endothelial cells, smooth muscle cells, and adventitial fibroblast-like cells, in which GRP78 protein was specifically localized to the cisternae of the rough ER and perinuclear cisternae, but not to other organelles such as mitochondria or nuclei. Thus, our data provide novel insights into the phenotypic and functional heterogeneity of GRP78-positive cells within the lesion core, suggesting the involvement of GRP78 in the activation/recruitment of activated microglia/macrophages and its potential role in BBB impairment in response to a 3-NP-mediated neurotoxic insult.

Expression of the Endoplasmic Reticulum Stress Marker GRP78 in the Normal Retina and Retinal Degeneration Induced by Blue LED Stimuli in Mice

“…In this study, we demonstrated that GRP78 expression was increased in two types of retinal glial cells, Müller cells and microglial cells, in a blue LED-induced RD model. These results were consistent with the increase in GRP78 expression in glial cells in response to several types of brain and spinal cord injuries [25,[42][43][44]. Müller cells are the main glial cells in the retina, corresponding to astrocytes in the brain and spinal cord.…”

Osteopontin and its spatiotemporal relationship with glial cells in the striatum of rats treated with mitochondrial toxin 3-nitropropionic acid: possible involvement in phagocytosis

“…These Opn -expressing brain macrophages could have been derived either from resident microglia or from infiltrating blood-borne macrophages, as the two are indistinguishable by morphological criteria due to a lack of specific discrimination criteria [41–43]. Additionally, blood-brain barrier breakdown in the striatum has been reported in rat models of Huntington’s disease induced by 3-NP [35, 44, 45]. As noted above, some OPN-positive puncta were observed within the cytoplasm of Opn mRNA-expressing brain macrophages, corresponding to the Golgi complex, but most OPN-immunoreactive puncta were not associated with brain macrophages (Fig.…”

“…The mitochondrial toxin, 3-NP, irreversibly inhibits the mitochondrial respiratory chain complex II and impairs mitochondrial energy production [30, 31]. This toxin selectively damages medium-spiny striatal neurons and astrocytes in the well-demarcated lesion core, which triggers astroglial hypertrophy and the resultant astroglial scar formation in the peri-lesional area [16, 32–35]. In our previous study using the same model, we showed that the OPN protein is first localized within dendritic mitochondria, and then accumulates on the surface of degenerating dendrites during the first 2 weeks after 3-NP injection [16].…”

Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy

“…However, due to the pixel resolution limitations, the blurring of the fluorescent signals was a major difficulty in the correlation between LM imaging and EM imaging. FluoroNanogold (FNG) provides a precise correlation of the subcellular target proteins; for example, GRP-78-Bip is localized in the ER membranes in the EM images [ 16 , 17 , 18 , 19 , 20 ]. In addition, CLEM has been performed using consecutive sections of semithin and ultrathin sections, on which immunolabeling and LM observations were performed on the semithin sections and correlative electron microscopy was carried out on the consecutive ultrathin sections, which inevitably made the correlation of both images inaccurate [ 21 ].…”

“…In addition, CLEM has been performed using consecutive sections of semithin and ultrathin sections, on which immunolabeling and LM observations were performed on the semithin sections and correlative electron microscopy was carried out on the consecutive ultrathin sections, which inevitably made the correlation of both images inaccurate [ 21 ]. In our protocol, however, we obtained every ultrathin section from the semi-thin-immunolabeled sample; thus, a more precise localization of the target proteins was possible [ 16 , 17 , 20 , 22 ]. Moreover, conventional resin embedding and positive staining using heavy metals after light microscopy provided better image contrast and resolution than when using Tokuyasu’s technique, in which the negative staining obscures the image contrast and the absence of the resin embedding process limits the spatial resolution [ 11 ].…”