J Korean Neurosurg Soc.  2023 Jul;66(4):400-408. 10.3340/jkns.2022.0115.

Nicotinamide Mononucleotide Adenylyl Transferase 2 Inhibition Aggravates Neurological Damage after Traumatic Brain Injury in a Rat Model

• Gu X
• Ni H
• Kan X
• Chen C
• Zhou Z
• Ding Z
• Li D
• Liu B

• KMID: 2543531
• DOI: http://doi.org/10.3340/jkns.2022.0115

Abstract

Objective
: Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a crucial factor for the survival of neuron. The role of NMNAT2 in damage following traumatic brain injury (TBI) remains unknown. This study was designed to investigate the role of NMNAT2 in TBI-induced neuronal degeneration and neurological deficits in rats.
Methods
: The TBI model was established in Sprague-Dawley rats by a weight-dropping method. Real-time polymerase chain reaction, western blot, immunofluorescence, Fluoro-Jade C staining, and neurological score analyses were carried out.
Results
: NMNAT2 mRNA and protein levels were increased in the injured-side cortex at 6 hours and peaked 12 hours after TBI. Knocking down NMNAT2 with an injection of small interfering RNA in lateral ventricle significantly exacerbated neuronal degeneration and neurological deficits after TBI, which were accompanied by increased expression of BCL-2-associated X protein (Bax).
Conclusion
: NMNAT2 expression is increased and NMNAT2 exhibits neuroprotective activity in the early stages after TBI, and Bax signaling pathway may be involved in the process. Thus, NMNAT2 is likely to be an important target to prevent secondary damage following TBI.

Keyword

Nicotinamide nucleotide adenylyltransferase 2; Traumatic brain injury; BCL2 associated X; Neurological deficit; Neuronal degenerative

Figure

• 

  Fig. 1. establishment of the TBI model and experimental design. A : Brains of sham and TBI rats, the area surrounding the contusion cortex area was assessed. B : experiment I was designed to detect the time course of NMNAT2 expression after TBI. C : experiment II was designed to investigate the role of NMNAT2 after TBI. TBI : traumatic brain injury, Sd : Sprague-dawley, Ctrl : control, PCR : polymerase chain reaction, NMNAT2 : nicotinamide mononucleotide adenylyl transferase 2, Bax : BCL-2-associated X protein, fJC : fluoro-Jade C.

• 

  Fig. 2. A : Amplification and melting temperature curves of NMNAT2 and GAPdH were obtained to evaluate the cycle thresholds and verify the specificity of real-time PCR amplification. These different colored lines represent different tissue samples. B : The relative mRNA expression levels of NMNAT2 were estimated by using the ratio of the number of target mRNAs to GAPdH mRNA in the sham and TBI groups at 6, 12, 24, 48, and 72 hours. The relative levels were normalized to the sham group. C : endogenous NMNAT2 protein expression was detected by western blot in the sham and TBI groups at 6, 12, 24, 48, and 72 hours. d : Quantification of the expression for NMNAT2 protein. The relative densities were normalized to the sham group. e : double immunofluorescence analysis of brain tissue using antibodies against NMNAT2 (green) and NeuN (red); nuclei were labeled with dAPI (blue) and the secondary antibodies (1 : 800 Alexa fluor 488 donkey anti-rabbit immunoglobulin [Ig] G anti-body, and Alexa fluor 555 donkey anti-mouse IgG antibody; Invitrogen, Carlsbad, CA, USA) (×400). Scale bar, 50 μm. f : The fluorescence intensities of NMNAT2 were detected in sham and TBI groups at 12 hours. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s post hoc test and Student’s t test. n=6 for each group. data are expressed as mean±standard error of mean. *p<0.05, **p<0.01, ***p<0.001 vs. sham group. NMNAT2 : nicotinamide mononucleotide adenylyl transferase 2, GAPdH : glyceraldehyde 3-phosphate dehydrogenase, TBI : traumatic brain injury, dAPI : 4’,6-diamidino-2-phenylindole dihydrochloride.

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  Fig. 3. NMNAT2 inhibition exacerbated TBI-induced neuronal degeneration and neurological deficits via Bax signaling. A : NMNAT2 and Bax expression were detected by western blot in the sham, TBI, TBI + control siRNA, and TBI + NMNAT2 siRNA. B and C : Quantification of NMNAT2 and Bax expression with relative levels normalized to the sham group. d : Representative of neurological behavior scores in each group at 72-hour post-TBI. e and f : Neuronal degeneration after TBI measured by fJC staining (×400) in the peri-injury cerebral cortex, quantified as fJC-positive cells/mm2. Scale bar, 50 μm. Statistical analyses were performed using Student’s t test. n=6 for each group. data are expressed as mean±standard error of mean. — : TBI; Ctrl : TBI + control-siRNA; siRNA : TBI + NMNAT2-siRNA. *p<0.05, **p<0.01, ***p<0.001 vs. sham group. &p<0.05 vs. TBI + control siRNA group. TBI : traumatic brain injury, siRNA : small intefering RNA, NMNAT2 : nicotinamide mononucleotide adenylyl transferase 2, Bax : BCL-2-associated X protein, ns : not significant, fJC : fluoro-Jade C.

Reference

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