ATF3 is a neuron-specific biomarker for spinal cord injury and ischaemic stroke

Abstract

Background: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions.

Methods: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits.

Results: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function.

Conclusions: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties.

Highlights: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.

Keywords: activating transcription factor 3 (ATF3); biomarker; neuronal injury; neuroprotection; spinal cord injury; stroke.

FIGURE 1

RNA‐Seq of mouse spinal cord after spinal cord injury. (a) Volcano plots of mouse spinal cord RNA‐sequencing (RNA‐Seq) results showing that Atf3, highlighted in purple, is one of the most significantly upregulated genes 4 h after spinal cord injury (SCI) (adjusted Benjamini‒Hochberg false discovery rate [BHFDR] p < .05). (b) Top 20 in Gene Ontology (GO) analysis of differentially expressed genes (DEG) from RNA‐Seq, showing multiple major pathways including mitogen‐activated protein kinase (MAPK) cascade, positive regulation of cell death, and regulation of extrinsic apoptotic signalling pathway, and negative regulation of phosphorus metabolic pathway.

FIGURE 2

Activating transcription factor 3 (ATF3) induction in the neurons of injured hemi‐cord after spinal cord injury (SCI). (a) Quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) confirms the remarkably increased Atf3 gene expression in mouse spinal cord 4 h after SCI. The results are normalized to Atf3 expression in control animals. Data are presented as mean ± SEM and were analysed with unpaired two‐tailed t‐test, ^*** p < .001, n = 3 in each group. (b) Representative immunohistochemical staining of ATF3 and NeuN in control and injured hemi‐cord 1 day after SCI. All ATF3⁺ cells are NeuN⁺ 1 day after SCI. Scale bar = 40 µm. The images are the magnification of the squared areas in Figure S1.

FIGURE 3

Activating transcription factor 3 (ATF3) is induced in the neurons in peri‐infarct area after ischaemic stroke. (a) Representative immunohistochemical staining of NeuN and ATF3 in control and peri‐infarct ischaemia region 1 day after permanent distal middle cerebral artery occlusion (pMCAO) in mice. All ATF3⁺ cells are NeuN⁺. (b) Representative immunohistochemical staining of ATF3 and Fluoro‐Jade C (FJC, a known marker for degenerating neurons) in the peri‐infarct ischemia region 1 day after pMCAO in mice. All FJC⁺ cells are ATF3⁺ (arrowheads), but some ATF3⁺ cells are FJC⁻ (arrows). Scale bar = 50 µm.

FIGURE 4

Increased plasma activating transcription factor 3 (ATF3) protein levels after rodent spinal cord injury (SCI) or ischaemic stroke. Enzyme‐linked immune‐sorbent assay (ELISA) results showing ATF3 protein level was detectable in mouse plasma, and its level was increased significantly post‐SCI (a) or ischaemic stroke (b). Data are presented as mean ± SEM and are analysed with unpaired two‐tailed t‐test, ^*** p < .001, n = 4−7 in each group.

FIGURE 5

Serum activating transcription factor 3 (ATF3) is elevated in clinical spinal cord injury (SCI) and ischaemic stroke patients 24 h after injury. (a) Human serum ATF3 levels were measured using a commercially available enzyme‐linked immune‐sorbent assay (ELISA) kit in healthy control (n = 7), trauma control patients without SCI or traumatic brain injury (TBI) 24 h after injury (n = 7), and SCI patients 24 h after injury (n = 30). Serum ATF3 levels in SCI patients were significantly higher than those in healthy control and trauma control patients, with no statistical difference between healthy control and trauma control groups. (b) The serum ATF3 levels in patients with different severity of SCI. American Spinal Injury Association Impairment Scale (AIS) D represents mild SCI, while AIS A indicates the most severe SCI. (c) Human serum ATF3 levels were measured by ELISA from non‐stroke patient controls (n = 8) and patients within 24 h of ischaemic stroke (n = 21). Serum ATF3 levels were significantly elevated in stroke patients. (d) The serum ATF3 levels in patients with different NIH Stroke Score/Scale (NIHSS) at 24 h. Data are presented as mean ± SEM and are analysed with one‐way analysis of variance (ANOVA) with Bonferroni's multiple comparison tests (a, b and d) or unpaired two‐tailed t‐test (c), ^**** p < .0001, ^*** p < .001, ^** p < .01, ^* p < .05 and ‘ns’ as not statistically significant.

FIGURE 6

Atf3 knockout (KO) mice had worse neurological outcomes in spinal cord injury (SCI) or ischaemic stroke models. (a) Paw placement in a cylinder task showing that Atf3 KO mice had worse functional recovery after SCI compared to wild‐type (WT) mice. Sticker removal time from right paw (b) and quantification of left turns in corner test (c) showing that Atf3 KO mice had more severe sensorimotor dysfunction than WT mice 3 days after left permanent distal middle cerebral artery occlusion (pMCAO). Data are presented as mean ± SEM, n = 7−12 in each group and are analysed with two‐way analysis of variance (ANOVA) and Sidak's multiple comparisons tests, ^**** p < .0001, ^*** p < .001 and ‘ns’ as not statistically significant.

FIGURE 7

Atf3 knockout (KO) mice had worse tissue injury after spinal cord injury (SCI) or ischaemic stroke. (a) SCI lesion area measured by Eriochrome cyanine (EC) staining, with the schematic outline, and (b) the quantification of the injury area in spinal cord of wild‐type (WT) and Atf3 KO mice 2 weeks after SCI. Scale bar = 200 µm. The injury size, presented as the percentage of ipsilateral lesion area in total contralateral uninjured area, was larger in Atf3 KO mice than WT mice 2 weeks post‐SCI. n = 6 or 7 in each group. Representative images of cresyl violet‐stained serial brain sections 3 days after permanent distal middle cerebral artery occlusion (pMCAO) (c) and their quantification (d) showing that Atf3 KO mice had larger infarct volume than WT mice. Scale bar = 1 mm. n = 6 in each group. (e) Atf3 KO mice had increased numbers of FJC⁺ degenerating neurons 3 days after stroke. n = 6 in each group. Data are presented as mean ± SEM and are analysed with unpaired two‐tailed t‐test, ^*** p < .001.