The Neuroprotective Role of Myeloid Zinc Finger‐1 and Specificity Protein 1 during Treatment with Leukemia Inhibitory Factor
Authors: Davis Stephanie , Collier Lisa , Fazal Jawad , Britton Michael , Leonardo Christopher , Ajmo Craig , Pennypacker Keith 
Published: November 2023

Objective

The objective of this study is to determine whether leukemia inhibitory factor (LIF) induces neuroprotection through the transcription factors myeloid zinc finger‐1 (MZF‐1) and specificity protein 1 (Sp1).

Methods

After middle cerebral artery occlusion or sham surgery, male Sprague‐Dawley rats were injected with PBS or LIF (125 μg/kg) (n=4 per group). Rats were euthanized 72 h after injury. Western blotting was used to measure MZF‐1 and Sp1 protein expression in brain tissue and spleen tissue. Genomatix was used to identify MZF‐1 and Sp1 binding sites in the promoter of superoxide dismutase 3 (SOD3), a LIF‐inducible gene. Gel shift assays were used to confirm binding of these transcription factors in brain nuclear extracts. Fluorescent immunohistochemistry was used to visualize localization of Sp1, MZF‐1, and SOD3. For in vitro studies, cultured rat neurons were isolated at embryonic day 18 and transfected with scrambled or MZF‐1 siRNA (n=3 per group). Neurons were treated with PBS or 200 ng/mL LIF prior to 24 h in vitro ischemia induced by oxygen glucose deprivation. Lactate dehydrogenase (LDH) release was measured to assess neuronal death. SOD3, Sp1, and MZF‐1 mRNA levels were measured with real‐time PCR. MZF‐1 protein levels were quantified in cultured neurons with immunocytochemistry.

Results

LIF treatment did not significantly alter MZF‐1 or Sp1 expression in the brain at 72 h after MCAO. However, LIF significantly increased expression of MZF‐1 in the spleen at 72 h post‐MCAO compared to the PBS treatment. Four MZF‐1 binding sites and two Sp1 binding sites were identified in the rat SOD3 promoter using Genomatix, and confirmed using gel shift assays. LIF treatment caused nuclear accumulation of MZF‐1 at 72 h after MCAO, while Sp1 and MZF‐1 co‐localized with SOD3 at this time point. In cultured neurons, LIF treatment prior to 24 h in vitro ischemia significantly increased the percentage of MZF‐1‐positive neurons compared to PBS treatment (p<0.01). Real time PCR confirmed the increase in MZF‐1 mRNA LIF‐treated neurons compared to PBS‐treated neurons (p<0.05). In these same samples, LIF increased SOD3 mRNA after 24 h ischemia compared to PBS. Sp1 mRNA was not significantly altered by LIF treatment after 24 h ischemia. However, transfection of cultured neurons with MZF‐1 and Sp1 siRNA counteracted the significant decrease in LDH release observed after LIF treatment alone (p<0.05).

Conclusions

LIF induces neuroprotection against ischemia through the transcription factors MZF‐1 and Sp1. Moreover, the LIF‐mediated increase in MZF‐1 activity occurs through increased gene and protein expression in cultured neurons as well as splenic tissue

Support or Funding Information

Funding for experiments in this study was provided for by the National Institute for Neurological Disorders and Stroke (project numbers 1R56NS091146‐01, 7R01NS091146‐02, and 1R01NS091146‐01Al),