6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase Suppresses Neuronal Apoptosis by Increasing Glycolysis and "cyclin-dependent kinase 1-Mediated Phosphorylation of p27 After Traumatic Spinal Cord Injury in Rats

Abstract

Apoptosis is a vital pathological factor that accounts for the poor prognosis of traumatic spinal cord injury (t-SCI). The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is a critical regulator for energy metabolism and proven to have antiapoptotic effects. This study aimed to investigate the neuroprotective role of PFKFB3 in t-SCI. A compressive clip was introduced to establish the t-SCI model. Herein, we identified that PFKFB3 was extensively distributed in neurons, and PFKFB3 levels significantly increased and peaked 24 h after t-SCI. Additionally, knockdown of PFKFB3 inhibited glycolysis, accompanied by aggravated neuronal apoptosis and white matter injury, while pharmacological activation of PFKFB3 with meclizine significantly enhanced glycolysis, attenuated t-SCI-induced spinal cord injury, and alleviated neurological impairment. The PFKFB3 agonist, meclizine, activated cyclin-dependent kinase 1 (CDK1) and promoted the phosphorylation of p27, ultimately suppressing neuronal apoptosis. However, the neuroprotective effects of meclizine against t-SCI were abolished by the CDK1 antagonist, RO3306. In summary, our data demonstrated that PFKFB3 contributes robust neuroprotection against t-SCI by enhancing glycolysis and modulating CDK1-related antiapoptotic signals. Moreover, targeting PFKFB3 may be a novel and promising therapeutic strategy for t-SCI.

Keywords: PFKFB3; glycolysis; meclizine; neuronal apoptosis; traumatic spinal cord injury.

Fig. 1.

Experimental designs (A). The time course of PFKFB3, and F2,6BP showed that the levels of PFKFB3 and F2,6BP were significantly elevated at 6 h, peaked at 24 h, and then significantly decreased after 24 h postinjury. Representative western blot images (B); the levels of PFKFB3 (C); and the levels of F2,6BP (D). The expression of FBKFB3 in neurons was increased at 24 h postinjury. Representative microphotographs of double immunofluorescence of PFKFB3 and NeuN (E); the proportion of PFKFB3-positive neurons (F). N = 6 for each group. Data are expressed as the mean ± SD. *P < 0.05 versus sham; ^# P < 0.05 versus t-SCI 24 h. F2,6BP: fructose-2,6-bisphosphate; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; SD: standard deviation.

Fig. 2.

Knockdown of PFKFB3 decreased F2,6BP and lactic acid, while increased apoptosis and white matter injury at 24 h postinjury. Representative western blot images (A); the levels of PFKFB3 (B); the levels of F2,6BP (C); the Bcl-2/Bax ratio (D); the levels of CC-3 (E); the lactic acid levels (F); the levels of MBP (G); the levels of APP (H). N = 6 for each group. Data are expressed as the mean ± SD. *P < 0.05 versus sham; ^# P < 0.05 versus t-SCI + vehicle; ^@ P < 0.05 versus t-SCI + scramble siRNA. F2,6BP: fructose-2,6-bisphosphate; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; SD: standard deviation; siRNA: small interfering RNA; t-SCI: traumatic spinal cord injury.

Fig. 3.

Meclizine increased PFKFB3, F2,6BP, and lactic acid, while decreased apoptosis and white matter injury at 24 h postinjury. Representative western blot images (A); the levels of PFKFB3 (B); the levels of F2,6BP (C); the Bcl-2/Bax ratio (D); the levels of CC-3 (E); the lactic acid levels (F); the levels of MBP (G); the levels of APP (H). N = 6 for each group. Data are expressed as the mean ± SD. *P < 0.05 versus sham; ^# P < 0.05 versus t-SCI + vehicle; ^@ P < 0.05 versus t-SCI + meclizine. F2,6BP: fructose-2,6-bisphosphate; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; SD: standard deviation; t-SCI: traumatic spinal cord injury.

Fig. 4.

Meclizine improved motor function of t-SCI rats. The BBB scores (A); the CBS (B). N = 6 for each group. Data are expressed as the mean ± SD. *P < 0.05 versus sham; ^# P < 0.05 versus t-SCI + vehicle. BBB: Basso, Beattie, and Bresnahan; dpi: day postinjury; SD: standard deviation; t-SCI: traumatic spinal cord injury.

Fig. 5.

Meclizine’s antiapoptotic effects were mediated by CDK1 and could be reversed by RO3306 at 24 h postinjury. Representative western blot images (A); the levels of nuc-PFKFB3 (B); the levels of nuc-F2,6BP (C); the CDK1 activities (D); the levels of nuc-p-CDK1 (E); the nuc-p-p27/nuc-p27 ratio (F); the Bcl-2/Bax ratio (G); and the levels of CC-3 (H). N = 6 for each group. Data are expressed as the mean ± SD. *P < 0.05 versus t-SCI + vehicle; ^# P < 0.05 versus t-SCI + meclizine; ^@ P < 0.05 versus t-SCI + RO3306. CDK1: cyclin-dependent kinase 1; F2,6BP: fructose-2,6-bisphosphate; PFKFB3: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; SD: standard deviation; t-SCI: traumatic spinal cord injury.

Fig. 6.

Meclizine’s antiapoptotic effects were reversed by RO3306 at 24 h postinjury. Representative microphotographs of double immunofluorescence staining of CC-3 and NeuN (A); the proportion of CC-3-positive neurons (B). Data are expressed as mean ± SD. *P < 0.05 versus t-SCI + vehicle; ^# P < 0.05 versus t-SCI + meclizine; ^@ P < 0.05 versus t-SCI + RO3306. SD: standard deviation; t-SCI: traumatic spinal cord injury.

Fig. 7.

Meclizine’s antiapoptotic effects were reversed by RO3306 at 24 h postinjury. Representative microphotographs of double immunofluorescence staining of TUNEL and NeuN (A); the proportion of TUNEL-positive neurons (B). N = 6 for each group. Data are expressed as mean ± SD. *P < 0.05 versus t-SCI + vehicle; ^# P < 0.05 versus t-SCI + meclizine; ^@ P < 0.05 versus t-SCI + RO3306. SD: standard deviation; t-SCI: traumatic spinal cord injury; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Fig. 8.

Meclizine’s antiapoptotic effects were reversed by RO3306 at 24 h postinjury. Representative microphotographs of TEM showing nuclei (diamonds), mitochondria (×4,200, arrow), and mitochondria (×11,500, double arrow) (A); the proportion of mitochondrial vacuolization (B). N = 6 for each group. Data are expressed as the mean ± SD. *P < 0.05 versus t-SCI + vehicle; ^# P < 0.05 versus t-SCI + meclizine; ^@ P < 0.05 versus t-SCI + RO3306. SD: standard deviation; t-SCI: traumatic spinal cord injury; TEM: transmission electron microscopy.

Fig. 9.

The signaling map showed the potential molecular mechanisms of meclizine’s antiapoptotic effects.