FAM3A Ameliorates Brain Impairment Induced by Hypoxia-Ischemia in Neonatal Rat

Abstract

Hypoxia-ischemia (HI) during crucial periods of brain formation can lead to changes in brain morphology, propagation of neuronal stimuli, and permanent neurodevelopmental impairment, which can have profound effects on cognitive function later in life. FAM3A, a subgroup of family with sequence similarity 3 (FAM3) gene family, is ubiquitously expressed in almost all cells. Overexpression of FAM3A has been evidenced to reduce hyperglycemia via the PI3K/Akt signaling pathway and protect mitochondrial function in neuronal HT22 cells. This study aims to evaluate the protective role of FAM3A in HI-induced brain impairment. Experimentally, maternal rats underwent uterine artery bilateral ligation to induce neonatal HI on day 14 of gestation. At 6 weeks of age, cognitive development assessments including NSS, wire grip, and water maze were carried out. The animals were then sacrificed to assess cerebral mitochondrial function as well as levels of FAM3A, TNF-α and IFN-γ. Results suggest that HI significantly reduced FAM3A expression in rat brain tissues, and that overexpression of FAM3A through lentiviral transduction effectively improved cognitive and motor functions in HI rats as reflected by improved NSS evaluation, cerebral water content, limb strength, as well as spatial learning and memory. At the molecular level, overexpression of FAM3A was able to promote ATP production, balance mitochondrial membrane potential, and reduce levels of pro-inflammatory cytokines TNF-α and IFN-γ. We conclude that FAM3A overexpression may have a protective effect on neuron morphology, cerebral mitochondrial as well as cognitive function. Created with Biorender.com.

Keywords: Brain; FAM3A; Hypoxia–ischemia; IFN-γ; Mitochondria; TNF-α.

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Fig. 1

HI inhibits FAM3A expression and contributes to increased mortality rate. A survival rate of pups born in the control were significantly higher than that of the HI group. B mRNA level of FAM3A in brain tissues show decreased FAM3A expression, lentivirus transduction showed increased FAM3A expression. C and D image and quantification of FAM3A protein expression. E and F image and quantification of immunofluorescence of FAM3A in CA1 using confocal microscope. n = 6 animals from each group 6 fields were taken for 3 slices per animal, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 2

FAM3A expression contributes to improved neuronal cell morphology. A neuronal cell morphology in the cortex and hippocampus regions of the brain were examined through H&E staining. Neuronal cells in the HI group showed dark staining and small pyknotic nuclei, while control and FAM3A overexpressing groups demonstrated normal and round nuclei. Original magnification: ×400. B and C number of non-damaged neurons in the cortex and hippocampus. HI-LV-Ctrl group shows significantly less number of non-damaged cells compared to the Ctrl-LV-Ctrl and HI-LV-FAM3A groups. D % of cerebral water content. HI group demonstrated a 2.3% increase in cerebral water content, while FAM3A overexpression led to a 1.3% reduction. n = 6 animals from each group, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 3

The effect of HI and FAM3A expression on memory, spatial learning, and motor function. A cognitive function assessments were assessed when the animals were 6.5 weeks-old. NSS evaluation was based on 10-point system, with high scores indicative of more severe neurological impairments. NSS values for Ctrl-LV-Ctrl, HI-LV-Ctrl, and HI-LV-FAM3A are 0, 2.83, and 1.5, respectively. B and C grip test was performed at 6.5 weeks, both scores and grip time were recorded. Both overall grip score and amount of time spent holding onto the wire were significantly less in the HI group compared to the control group. HI-LV-FAM3A rats performed significantly better than the HI-LV-Ctrl rats on grip score and grip time. D the escape latency period in the Morris water maze test showed that rats in the HI group required more time to escape platform compared to the control group, and that HI-LV-FAM3A group required less time compared to the HI-LV-Ctrl group. n = 6 animals from each group, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 4

Swimming tracts for the Morris Water Maze test. Representative swimming tracks in the Morris water maze for days 1–5

Fig. 5

HI inhibits while FAM3A enhances mitochondrial function. A membrane potential was assessed using JC-1 dye. The percentage reflects the ratio of JC-1 aggregates and JC-1 molecules, the higher the percentage is indicative of lower membrane potential. The HI-LV-Ctrl group exhibited lower membrane potential compared to Ctrl-LV-Ctrl and HI-LV-FAM3A groups. B–E activities of mitochondrial complexes I-IV (NADH-coenzyme Q reductase, succinate-coenzyme Q reductase, ubiquinol-cytochrome C reductase, and cytochrome C oxidase) were reduced in HI rats, and FAM3A expression did not significantly promote their activity levels. F ATP level in the HI-LV-Ctrl group was significantly lower than that of the Ctrl-LV-Ctrl and HI-LV-FAM3A group. G and H ROS levels and SOD activity were higher in the HI-LV-Ctrl group compared to the Ctrl-LV-Ctrl group, but not significantly different from HI-LV-FAM3A group. I and J ELISA results show heightened TNF-α and IFN-γ levels in the HI-LV-Ctrl group compared to the Ctrl-LV-Ctrl as well as the HI-LV-FAM3A group. n = 6 animals from each group, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001