Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen-glucose deprivation

Abstract

Carboxy terminus of Hsc70-interacting protein (CHIP) is thought to be a cytoprotective protein with protein quality control roles in neurodegenerative diseases and myocardial ischemia. This study describes the localization of CHIP expression in normal rodent brain and the early CHIP response in primary cultures of cortical neurons following ischemic stress models: heat stress (HS) and oxygen-glucose deprivation (OGD). CHIP was highly expressed throughout the brain, predominantly in neurons. The staining pattern was primarily cytoplasmic, although small amounts were seen in the nucleus. More intense nuclear staining was observed in primary cultured neurons which increased with stress. Nuclear accumulation of CHIP occurred within 5-10 min of HS and decreased to baseline levels or lower by 30-60 min. Decrease in nuclear CHIP at 30-60 min of HS was associated with a sharp increase in delayed cell death. While no changes in cytoplasmic CHIP were observed immediately following OGD, nuclear levels of CHIP increased slightly in response to OGD durations of 30 to 240 min. OGD-induced increases in nuclear CHIP decreased slowly during post-ischemic recovery. Nuclear CHIP decreased earlier in recovery following 120 min of OGD (4 h) than 30 min of OGD (12 h). Significant cell death first appeared between 12 and 24 h after OGD, again suggesting that delayed cell death follows closely behind the disappearance of nuclear CHIP. The ability of CHIP to translocate to and accumulate in the nucleus may be a limiting variable that determines how effectively cells respond to external stressors to facilitate cell survival. Using primary neuronal cell cultures, we were able to demonstrate rapid translocation of CHIP to the nucleus within minutes of heat stress and oxygen-glucose deprivation. An inverse relationship between nuclear CHIP and delayed cell death at 24 h suggests that the decrease in nuclear CHIP following extreme stress is linked to delayed cell death. Our findings of acute changes in subcellular localization of CHIP in response to cellular stress suggest that cellular changes that occur shortly after exposure to stress ultimately impact on the capacity and capability of a cell to recover and survive.

Fig. 1

CHIP immunoreactivity in brain sections from adult male Long-Evans rats (a). Coronal sections were taken from the cortical and ependymal cell regions. The sections were stained with anti-CHIP antibody (green) and anti-MAP2 antibody (red) along with the nuclear counterstain bisbenzimide (blue). Merged images are shown in panels on the far right. CHIP immunocytochemical staining of cultured neurons shows reactivity in both neurons and astrocytes (b). Cultured neurons (10 days in vitro) were stained for CHIP (green) and MAP-2 (red) along with nuclear counterstain, bisbenzimide (blue). A merged image is shown in the panel on the far right

Fig. 2

CHIP immunoreactivity in brain sections from adult male Sv129 mice. Immunoreactivity in the mouse brain was observed in the cortex (a), hippocampus (b), hypothalamus (c), Purkinje neurons (d), choroid plexus and ependymal cells (e), and thalamus (f). The lack of co-localization of cortical CHIP staining (g) and the nuclear counterstain bisbenzimide (h) in a merged image (i) suggests a primarily cytoplasmic localization of CHIP

Fig. 3

Total CHIP levels did not change immediately following increasing duration of heat stress (a). Equal protein concentrations from whole-cell lysate of primary mixed cultures exposed to heat stress were probed for CHIP. β-actin is shown as a loading control. Nuclear CHIP decreases following extended durations of heat stress (b). Equal protein concentrations from subcellular fractions of primary mixed cultures exposed to heat stress were probed for CHIP, HSC70, and HSP70 by Western blot. β-actin is shown as a loading control

Fig. 4

Cell death after prolonged heat stress is increased after a 24-h recovery period. Cultures subjected to durations of heat stress between 0 and 60 min, recovered in normal culture conditions for 24 h and stained with Sytox Green (1 µM). Sytox Green intensity was measure by plate reader. Relative intensity was analyzed as percent increase over baseline ± SEM at 24 h of recovery after 5 (55.45% ± 3.51), 10 (74.85 ± 6.35), 15 (115.78% ± 8.36), 30 (198.3% ± 12.14), and 60 min (735.41% ± 76.84) of heat stress. Measurements following 30 and 60 min were significant (*p < .001, n = 47)

Fig. 5

Cell death after increasing periods of OGD and a 24-h recovery period. Cultures subjected to durations of OGD between 0 and 240 min, recovered in normal culture conditions for 24 h and stained with Sytox Green (1 µM). Sytox Green intensity was measured by plate reader. Sytox Green intensity was analyzed as percent increase over baseline 24 h (a) after 30 (23.06% ± 3.75), 60 (58.95 ± 4.48), 120 (63.36% ± 5.32), and 240 min (123.74% ± 8.62) of OGD. Measurements following 60 to 240 min were significant (*p < .001, n = 47). Sytox Green-positive cells were analyzed as a percentage of all cells in culture after the 24-h recovery period (b) following 0 (11.6% ± 0.113), 30 (25.3% ± 0.194), 60 (33.8% ± 0.167), 120 (45.7% ± 0.166), and 240 min (88.3% ± 0.113) of OGD *p < .001, **p < .05

Fig. 6

Relative amounts of subcellular CHIP are slightly altered immediately following OGD (a). Total CHIP levels did not change significantly during recovery following 30 and 120 minutes of OGD (b). Equal protein concentrations from whole-cell lysate of primary mixed cultures were probed for CHIP. Equal protein concentrations from subcellular fractions of primary mixed cultures exposed to OGD were probed for CHIP by Western blot (c). No changes are observed in the cytoplasmic fraction with increasing durations of OGD. In the nuclear fraction, a slight increase is observed following 30 min of OGD is sustained, but not otherwise altered following increasing durations of OGD. Elevated levels of nuclear CHIP are maintained during recovery after 30 min of OGD. Equal protein concentrations from subcellular fractions of primary mixed cultures exposed to mild (30 min) and severe (120 min) OGD were probed for CHIP, HSC70, and HSP70 by Western blot