Ethanol withdrawal acts as an age-specific stressor to activate cerebellar p38 kinase

Abstract

We investigated whether protein kinase p38 plays a role in the brain-aging changes associated with repeated ethanol withdrawal (EW). Ovariectomized young, middle-age and older rats, with or without 17β-estradiol (E2) implantation, received a 90-day ethanol with repeated withdrawal. They were tested for active pP38 expression in cerebellar Purkinje neurons and whole-cerebellar lysates using immunohistochemistry and enzyme-linked immunosorbent assay, respectively. They were also tested for the Rotarod task to determine the behavioral manifestation of cerebellar neuronal stress and for reactive oxygen species (ROS) and mitochondrial protein carbonyls to determine oxidative mechanisms. Middle-age EW rats showed higher levels of pP38-positive Purkinje neurons/cerebellar lysates, which coincided with increased mitochondrial protein oxidation than other diet/age groups. Exacerbated motor deficit due to age-EW combination also began at the middle-age. In comparison, ROS contents peaked in older EW rats. E2 treatment mitigated each of the EW effects to a different extent. Collectively, pP38 may mediate the brain-aging changes associated with pro-oxidant EW at vulnerable ages and in vulnerable neurons in a manner protected by estrogen.

Figure 1.. EW-induced P38 activation.

The protein levels of cerebellar pP38 were assessed using an immunoblot method 2 weeks after a 5-week ethanol diet (6.5% v/v) in young adult male rats. The levels of pP38 were much higher during EW than during ethanol-exposure, but the levels of total P38 did not significantly differ between diet groups. ^*p < 0.01, ^**p < 0.001 vs. a control dextrin (=100%). Depicted are mean ± SEM for 3 rats/group.

Figure 2.. Cytotoxic P38 activation.

The effect of EW on HT22 cell viability was measured 4 hours after 24-hour ethanol exposure. EW decreased cell viability relative to control cell viability at 100% (statistical symbols were omitted to simplify figures). SB203580 (200 nM) treatment during the EW phase (right), but not the ethanol-exposure phase (left), mitigated EW-induced cell death. ^*p = 0.039, ^**p = 0.009 vs. vehicle (non SB203580). Depicted are mean ± SEM for 4/treatment.

Figure 3.. Age-dependent motor deficit in ethanol-withdrawn rats.

Ovariectomized rats were implanted with oil or E2 pellets, received a 90-day diet regimen, a 25-day ethanol- (6.5% v/v) or a control dextrin diet followed by 5-day abrupt withdrawal, and the cycle was repeated 3 times. Beginning at the 7th day of EW, the rats were tested for the Rotarod task (3 sessions/day, 5 days). Data were presented as an average latency from the 5-day data. Motor deficit (shorter latency) was found in young EW rats (p < 0.01) compared to other diet groups, became more severe (p < 0.01) in middle-age EW rats, and then remained at a similar level in older EW rats. ^*p < 0.01 vs. dextrin, dextrin + E2 or EW+E2 at each age. ^‡p < 0.05 vs. 8-month-old dextrin. ^†p < 0.01 vs. 8-month-old EW. Depicted are mean ± SEM for 4–7 rats/group.

Figure 4.. P38 activation in whole cerebellar extracts.

Ovariectomized rats underwent repeated EW as described in the Figure 3 legend. At the 14th day of EW, the protein level of pP38 was measured in the cerebellar whole-cell lysates using ELISA assay. The middle-age EW rats had a higher level of pP38 than other diet or age groups (left). E2 per se did not significantly alter the level of pP38 in dextrin diet rats (right). ^*p < 0.01 vs. all other groups. Depicted are mean ± SEM for 4 or 5 rats/group.

Figure 5.. pP38-positive Purkinje neurons.

Ovariectomized rats underwent repeated EW as described in the Figure 3 legend. At the 14th day of EW, the left hemisphere containing the cerebellar vermis was processed for immunohistochemical analysis. All photographs were taken of the cerebellar cortex area containing Purkinje layers that showed a clear image across all treatment groups. Dark deposits marked with arrows indicate pP38 immunoreactivity in Purkinje neurons along the Purkinje layer, and they distinctively appeared in the EW groups across all 3 ages (Figure 5A-D). EW rats had a higher number of pP38-positive Purkinje neurons/Purkinje layer (mm) than dextrin, ethanol or EW+E2 rats, particularly at 15 months old (Figure 5E). ^*p < 0.01 vs. dextrin or EW+E2 at 8 or 19 months old; ^**p < 0.01 vs. dextrin, Ethanol or E2 at 15 months old; ^†p < 0.01 vs. 8- or 19-month-old EW; ^‡p < 0.05 vs. 8-month-old-dextrin. A 5-fold (Figure 5A) or 20-fold (Figure 5B-D) magnification was used to take pictures of representative groups (15 months old) or all age/diet groups, respectively. Figure 5A shows cerebellar lobule III and IV and Figure 5B-D show lobule II. The scale bar indicates an actual length of 200 μm (Figure 5A) or 50 μm (Figure 5B-D). Depicted are mean ± SEM for 6 microscopic fields/rat for 3 rats/group (Figure 5E).

Figure 6.. pSTAT-positive Purkinje neurons.

Ovariectomized rats underwent repeated EW as described in the Figure 3 legend. At the 14th day of EW, the left hemisphere containing the cerebellar vermis was processed for immunohistochemical analysis to identify pSTAT1-positive Purkinje neurons. Photographs were taken of the cerebellar cortex of lobule II containing Purkinje layers of middle-age rats (15 months). Dark deposits marked with arrows indicate pSTAT1 immunoreactivity in Purkinje neurons, and they distinctively appeared in the EW group. EW rats had a higher number of pSTAT1-positive Purkinje neurons/Purkinje layer (mm) than dextrin (^*p = 0.005) or EW+E2 (^†p = 0.001) rats. A 20-fold magnification was used to take pictures. The scale bar indicates an actual length of 50 μm. Depicted are mean ± SEM for 6 microscopic fields/rat for 3 rats/group.

Figure 7.. Total ROS in rats and HT22 cells.

Ovariectomized rats underwent repeated EW as described in the Figure 3 legend (left). Within the same age groups, ROS content in whole-cerebellar lysates was higher in the EW group than in other diet groups. Between age groups, a higher ROS content was found in the older EW group than in the young (^**p < 0.01) or the middle-age (^*p < 0.05) EW groups. ^†p < 0.05 vs. young or middle-age ethanol; ^‡p < 0.05 vs. young or middle-age EW+E2. In HT22 cells (right), total ROS were measured at 4 hours of EW from 24-hour ethanol exposure (0–100 mM). SB203580 (200 nM) treatment during the EW phase but not the ethanol-exposure phase decreased EW-induced ROS generation. ^*p < 0.01, ^**p < 0.001 vs. non-ethanol control. ^†p < 0.01 vs. cells treated with SB203580 (200 nM) during ethanol-exposure at 50 mM or 100 mM of ethanol. Depicted are mean ± SEM for 5–7 animals/group or 4 cell plates/group.

Figure 8.. Mitochondrial protein oxidation.

Ovariectomized rats implanted with oil pellets underwent repeated EW as described in the Figure 3 legend. The carbonylation of mitochondrial proteins with molecular weights equal to or less than 130 KDa is shown in this picture. The EW group exhibited much stronger carbonyl signals (dark bands) than the dextrin groups especially in the middle-age rats at certain molecular weights, including those indicated with arrows. Cerebellar mitochondria were pooled from 5 rats/group.