Immunohistochemical analysis for acetylcholinesterase and choline acetyltransferase in mouse cerebral cortex after traumatic brain injury

Abstract

The regulation of glial cells, especially astrocytes and microglia, is important to prevent the exacerbation of a brain injury because over-reactive glial cells promote neuronal death. Acetylcholine (ACh), a neurotransmitter synthesized and hydrolyzed by choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively, in the central nervous system, has the potential to regulate glial cells' states, i.e., non-reactive and reactive states. However, the expression levels of these ACh-related enzymes in areas containing reactive glial cells are unclear. Herein we immunohistochemically investigated the distributions of AChE and ChAT with reactive glial cells in the cryo-injured brain of mice as a traumatic brain injury model. Immunohistochemistry revealed AChE-and ChATimmunopositive signals in injured areas at 7 days post-injury. The signals were observed in and around GFAP-or CD68-immunopositive cells, and the numbers of cells doubly positive for GFAP/AChE, GFAP/ChAT, CD68/AChE, and CD68/ChAT were significantly increased in injured areas compared to sham-operated areas. Enzyme histochemistry for AChE showed intensely positive signals in injured areas. These results suggest that reactive astrocytes and microglia express and secrete AChE and ChAT in brain-injury areas. These glial cells may adjust the ACh concentration around themselves through the regulation of the expression of ACh-related enzymes in order to control their reactive states.

Adiponectin-Receptor Agonistic Dipeptide Tyr–Pro Stimulates the Acetylcholine Nervous System in NE-4C Cells

“…As shown in Figures A–C and S2, treatment of NE-4C progenitor cells with 20 μM Tyr–Pro for 48 h caused a significant ( p < 0.05) increase in ChAT, a transferase enzyme responsible for ACh synthesis, similar to AdipoRon. In contrast, the expression of AChE, a cholinergic enzyme that can immediately hydrolyze ACh to acetate and choline, was not affected by Tyr–Pro or AdipoRon (Figures D and S2). No changes were observed in the m/nAChRs (Figures E,F, and S2).…”

“…25 Thus, an appropriate assay for ACh production may contribute to AD diagnostic evaluation, although ACh is liable to rapid enzymatic degradation by AChE in the brain tissue. 18 An electrochemical detector-assisted HPLC assay or a commercially available ELISA kit is commonly used to determine ACh, despite its poor selectivity and sensitivity. 26 Thus, in the present study, we proposed a LC-TOF/MS assay using a multimode-type column with hydrophobic and cationexchange characteristics combined with a surrogate compound, ACh-d 4 , as a novel ACh assay.…”

“…To confirm the brain-beneficial effect of Tyr−Pro in the ACh nervous system, the amount of ACh production in NE-4C nerve cells in the presence of 10 μM donepezil (AChE inhibitor) was analyzed using LC-ESI-TOF/MS on a multimode Scherzo SS-C18 column having hydrophobic and ionexchange characteristics. As shown in Figure 2A, an ACh compound capable of rapid degradation by AChE 18 18 similar to AdipoRon. In contrast, the expression of AChE, a cholinergic enzyme that can immediately hydrolyze ACh to acetate and choline, 18 was not affected by Tyr−Pro or AdipoRon (Figures 3D and S2).…”

“…As shown in Figure 2A, an ACh compound capable of rapid degradation by AChE 18 18 similar to AdipoRon. In contrast, the expression of AChE, a cholinergic enzyme that can immediately hydrolyze ACh to acetate and choline, 18 was not affected by Tyr−Pro or AdipoRon (Figures 3D and S2). No changes were observed in the m/nAChRs (Figures 3E,F, and S2).…”

Mass Spectrometric Enzyme Histochemistry for Choline Acetyltransferase Reveals De Novo Acetylcholine Synthesis in Rodent Brain and Spinal Cord

“…Choline acetyltransferase (ChAT; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6) is an essential enzyme responsible for the production of the neurotransmitter acetylcholine (ACh). ChAT is considered a specific marker for cholinergic neurons, and many studies have been conducted on its localization and activity together with acetylcholinesterase (AChE). − For instance, in-tissue visualization of ChAT has been performed using immunohistochemistry for the presence of the ChAT protein, , in situ hybridization for ChAT mRNA, , and generation of transgenic mice targeting the CHAT promoter . However, these techniques also encompass a few limitations; for example, immunohistochemistry might result in false positives, as it can recognize molecules with antigens similar to those of the ChAT protein .…”