Activation of the XBP1s/O-GlcNAcylation Pathway Improves Functional Outcome After Cardiac Arrest and Resuscitation in Young and Aged Mice

Abstract

After cardiac arrest (CA) and resuscitation, the unfolded protein response (UPR) is activated in various organs including the brain. However, the role of the UPR in CA outcome remains largely unknown. One UPR branch involves spliced X-box-binding protein-1 (XBP1s). Notably, XBP1s, a transcriptional factor, can upregulate expression of specific enzymes related to glucose metabolism, and subsequently boost O-linked β-N-acetylglucosamine modification (O-GlcNAcylation). The current study is focused on effects of the XBP1 UPR branch and its downstream O-GlcNAcylation on CA outcome. Using both loss-of-function and gain-of-function mouse genetic tools, we provide the first evidence that activation of the XBP1 UPR branch in the post-CA brain is neuroprotective. Specifically, neuron-specific Xbp1 knockout mice had worse CA outcome, while mice with neuron-specific expression of Xbp1s in the brain had better CA outcome. Since it has been shown that the protective role of the XBP1s signaling pathway under ischemic conditions is mediated by increasing O-GlcNAcylation, we then treated young mice with glucosamine, and found that functional deficits were mitigated on day 3 post CA. Finally, after confirming that glucosamine can boost O-GlcNAcylation in the aged brain, we subjected aged mice to 8 min CA, and then treated them with glucosamine. We found that glucosamine-treated aged mice performed significantly better in behavioral tests. Together, our data indicate that the XBP1s/O-GlcNAc pathway is a promising target for CA therapy.

Figure 1.. CA outcome was worse in young mice with Xbp1 deletion in forebrain neurons.

(A) Representative laser speckle contrast images showing cortical cerebral blood flow changes in our CA/CPR model. (B-E) Young Xbp1-cKO (cKO) and Xbp1^f/f littermates (control) were subjected to 8.5 minutes CA. Rotarod (B), spontaneous locomotor activity (traveled distance during a 10-minute test period in an open field test; C), and neurologic score (D) were evaluated on days 1 and 3 after CA. Body weight loss was evaluated on day 3 after CA (E). Data are presented as mean ± SEM or median (n = 10–11/group). *, p < 0.05; **, p < 0.01.

Figure 2.. CA outcome was improved in young mice with neuron-specific overexpression of Xbp1s.

Young XBP1s-TG (TG) and littermate control mice were subjected to 8.5 minutes CA followed by resuscitation. Body weight loss was evaluated on day 3 after CA (A). Neurologic scoring was performed on days 1 and 3 after CA (B). A total of 6 mice (4 control mice and 2 TG mice) died on day 2 or day 3, and were excluded from data analysis. Data are presented as mean ± SEM or median (n = 8/group). **, p < 0.01.

Figure 3.. Short-term CA outcome was improved in young mice after post-CA treatment with glucosamine.

Young C57Bl/6 mice were subjected to 8.5 minutes CA followed by resuscitation. After 1 hour and 1 day of reperfusion, mice were treated with saline (vehicle) or glucosamine. Rotarod performance (A) and neurologic score (B) were evaluated on days 1 and 3 after CA. Data are presented as mean ± SEM or median (n = 6–7/group). *, p < 0.05; **, p < 0.01.

Figure 4.. Age affected dynamics of cerebral blood flow (CBF) recovery after CA/CPR.

Young and aged mice were subjected to 8.5 or 8 minutes CA, respectively. During the entire procedure (except the CPR step), brain cortical CBF was monitored using laser speckle contrast imaging (LSCI). (A) Representative LSCI images. The regions for quantification were marked with dashed rectangles. (B) Quantitative CBF measurement of the cortical areas by LSCI at the indicated time points over the course of the CA/CPR procedure. Baseline was set at 100% for each mouse. CBF change at each time point was calculated by comparing to baseline. Data are presented as mean ± SEM (n = 4/group). **, p < 0.01; ***, p < 0.001.

Figure 5.. Long-term CA outcome was improved in aged mice after post-CA treatment with glucosamine.

(A) Glucosamine-induced increase in O-GlcNAcylation in aged brains. Aged C57Bl/6 mice were dosed with glucosamine (GlcN) or vehicle, and 3 hours later, brain cortex samples were collected for Western blotting. (B-E) Long-term CA outcome. Aged mice (n = 9–11/group) were subjected to 8 minutes CA. Glucosamine was dosed at 1 hours after ROSC, and then on day 1 after CA. CA outcome evaluations included neurologic score (B), spontaneous locomotor activity (open field test; C), and body weight loss on post-CA day 7 (D). (E) Survival rates over 7 days of observation. Of note, one mouse from the vehicle group appeared to have hemangioma, and was excluded from analysis. Data are presented as mean ± SEM or median. *, p < 0.05; **, p < 0.01.