Par-4 Inhibits Choline Uptake by Interacting with CHT1 and Reducing Its Incorporation on the Plasma Membrane

Abstract

CHT1 is a Na؉ -and Cl ؊ -dependent, hemicholinium-3 (HC-3)-sensitive, high affinity choline transporter. Par-4 (prostate apoptosis response-4) is a leucine zipper protein involved in neuronal degeneration and cholinergic signaling in Alzheimer's disease. We now report that Par-4 is a negative regulator of CHT1 choline uptake activity. Transfection of neural IMR-32 cells with human CHT1 conferred Na ؉ -dependent, HC-3-sensitive choline uptake that was effectively inhibited by cotransfection of Par-4. Mapping studies indicated that the C-terminal half of Par-4 was physically involved in interacting with CHT1, and the absence of Par-4⅐CHT1 complex formation precluded the loss of CHT1-mediated choline uptake induced by Par-4, indicating that Par-4⅐CHT1 complex formation is essential. Kinetic and cell-surface biotinylation assays showed that Par-4 inhibited CHT1-mediated choline uptake by reducing CHT1 expression in the plasma membrane without significantly altering the affinity of CHT1 for choline or HC-3. These results suggest that Par-4 is directly involved in regulating choline uptake by interacting with CHT1 and by reducing its incorporation on the cell surface.Cholinergic neurotransmission plays a major role in the regulation of many physiological functions (1-3). For example, basal forebrain cholinergic neurons are involved in learning and memory processes, and hypoactivity of the cholinergic system may be directly responsible for the cognitive deficit in Alzheimer's disease (AD) 1 (4 -8). Reductions in choline acetyltransferase (ChAT) activity and a profound loss of cholinergic neurons in the nucleus basalis magnocellularis have been observed in AD brain (5-12). High affinity choline uptake is generally believed to be the rate-limiting step in acetylcholine synthesis in cholinergic neurons and is essential for cholinergic transmission in the central nervous system (13-16). Reduced choline uptake has been implicated in degenerating neurons in AD (15, 17), although spared cholinergic nerve terminals in AD may show a compensatory increase in choline uptake (18,19).CHT1 is a Na ϩ -and Cl Ϫ -dependent, hemicholinium-3 (HC-3)-sensitive, high affinity choline transporter, and it is located specifically in cholinergic neurons (13). The human CHT1 (hCHT1) gene is ϳ25 kb in length, contains nine exons, and has been mapped to chromosome II at 20,21). The hCHT1 protein is composed of 580 amino acids with a predicted molecular mass of ϳ63 kDa. Sequence analysis of the hCHT1 protein predicts 13 transmembrane domains (13,20,21). Par-4 (prostate apoptosis response-4) is a leucine zipper protein that plays an important role in neuronal dysfunction and cell death in neurodegenerative disorders (22-28). Levels of Par-4 expression are significantly increased in cortical, hippocampal, and cholinergic neurons in in vivo and in vitro models of AD (24), and blockade of Par-4 expression prevents neuronal death (23,24,27,29). In addition, Par-4 contributes to apoptosis and aberrant production of amyloid ␤-peptide 1-42 in neural cells ...

The high-affinity choline transporter CHT1 is regulated by the ubiquitin ligase Nedd4-2

“…A few proteins have been reported to interact with CHT1 and regulate its trafficking. Par-4, which is involved in neuronal degeneration and Alzheimer's disease, has been reported to interact with CHT1 and reduce its expression on the cell surface (23). SEC14-like protein 1 (SEC14L1) has been reported to interact with CHT1 and with the vesicular acetylcholine transporter (VAChT) to decrease CHT1 activity (20).…”

Reduction in CHT1-mediated choline uptake in primary neurons from presenilin-1 M146V mutant knock-in mice

“…The increase in K d clearly suggest a significant decrease in CHT1's affinity to its binding ligand. Because HC-3 is hydrophilic and impermeable to cell membrane, the HC-3 binding activity represents the expression level of the transporter in the plasma membrane (Xie and Guo, 2004). The observation that there was no significant difference in B max values among neurons from the wild-type mice and those from the PS-1 M146V mutant animals suggests that levels of CHT1 protein in the plasma membrane was not significantly altered by the PS-1 mutation (Fig.…”

“…Biotinylated proteins were considered to be mostly expressed on the cell surface because they were greatly depleted of the intracellular marker actin (Fig. 1d, bottom panel) (Xie and Guo, 2004). As shown in Figure 1d (top panel) and Fig.…”

“…These results suggest that the cholinergic signaling can be affected by mutations in presenilin-1. Of importance, we recently found that Par-4, a pro-apoptotic protein that mediates the adverse effect of the mutant presenilin-1, may directly alter CHT1 mediated choline uptake (Xie et al, 2001;Xie and Guo, 2004). These results raised the possibility that choline uptake might be altered in the knockin mouse expressing the AD-linked PS-1 mutation.…”

“…[ 3 H] Choline uptake assays were performed essentially as previously described (Okuda et al, 2002;Xie and Guo, 2004). In brief, cells were washed twice with Krebs-Ringer's-HEPES buffer (NaCl 130 mM, KCl 1.3 mM, MgSO 4 , 1.2 mM, CaCl 2 2.2 mM, KH 2 PO 4 1.2 mM, glucose 10 mM, HEPES 10 mM, pH 7.4) and incubated at 37°C for 2 hour , followed by an additional incubation in Krebs-Ringer's-HEPES buffer containing 1 μM [methyl-3 H] choline chloride (Amersham Biosciences; 83 Ci/mmol) for 10 min.…”