Abstract
Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the C-terminal domain of cannabinoid 1 receptor (CB1R) and regulates CB1R activities. In this study, we made Tat-CRIP1a fusion proteins to enhance CRIP1a penetration into neurons and brain and to evaluate the function of CRIP1a in neuroprotection following oxidative stress in HT22 hippocampal cells and transient forebrain ischemia in gerbils. Purified exogenous Tat-CRIP1a was penetrated into HT22 cells in a time and concentration-dependent manner and prevented H2O2-induced reactive oxygen species formation, DNA fragmentation, and cell damage. Tat-CRIP1a fusion protein also ameliorated the reduction of 14-3-3η expression by H2O2 treatment in HT22 cells. Ischemia–reperfusion damage caused motor hyperactivity in the open field test of gerbils; however, the treatment of Tat-CRIP1a significantly reduced hyperactivity 1 day after ischemia. Four days after ischemia, the administration of Tat-CRIP1a restored the loss of pyramidal neurons and decreased reactive astrocytosis and microgliosis induced by ischemic damage in the hippocampal cornu Ammonis (CA)1 region. Ischemic damage decreased 14-3-3η expression in all hippocampal sub-regions 4 days after ischemia; however, the treatment of Tat-CRIP1 ameliorated the reduction of 14-3-3η expression. These results suggest that Tat-CRIP1a attenuates neuronal damage and hyperactivity induced by ischemic damage, and it restores normal expression levels of 14-3-3η protein in the hippocampus.
Highlights
Cerebral ischemia is one of the leading causes of death globally and results in a lower quality of life in survivors due to neurological disabilities [1,2]
To generate a Tat-His-PDIA3 fusion protein, the human Cannabinoid receptor-interacting protein 1a (CRIP1a) gene was fused to a Tat peptide expression vector, and the His-CRIP1a protein was manufactured without a Tat domain
Prominent Tat-His-CRIP1 protein and His-CRIP1a protein bands were found with about 1.6–1.7 kDa differences, which is the molecular weight for the Tat peptide, on Western blot using rabbit anti-polyhistidine antibody (Figure 1A)
Summary
Cerebral ischemia is one of the leading causes of death globally and results in a lower quality of life in survivors due to neurological disabilities [1,2]. Ischemia results in deficiencies in glucose and oxygen and leads to an ion imbalance and impairment of cellular homeostasis [3,4]. Excessive neuronal depolarization by ischemia causes excitotoxicity by releasing excitatory. Cells 2020, 9, 1827 neurotransmitters, and an over-release of calcium ions activates deleterious enzymatic reactions, which can result in neuronal death [5]. The cannabinoid 1 receptor (CB1R) is a G protein-coupled receptor and is detected in the excitatory presynaptic regions in the hippocampus [9,10].
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