Overexpression of the High Affinity Choline Transporter in Cortical Regions Affected by Alzheimer's Disease. Evidence from Rapid Autopsy Studies
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Overexpression of the high affinity choline transporter in cortical regions affected by Alzheimer's disease. Evidence from rapid autopsy studies. T A Slotkin, … , G Bissette, F J Seidler J Clin Invest. 1994;94(2):696-702. https://doi.org/10.1172/JCI117387. Research Article Find the latest version: https://jci.me/117387/pdf Overexpression of the High Affinity Choline Transporter in Cortical Regions Affected by Alzheimer's Disease Evidence from Rapid Autopsy Studies T. A. Slotkin,* C. B. Nemeroff,* G. Bissette,* and F. J. Seidler* *Depar.tments of Pharmacology and Psychiatry, Duke University Medical Center, Durham, North Carolina 27710; and *DepartIment of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia 30322 Abstract choline has rendered it virtually impossible to evaluate directly how the disease affects levels or turnover of this transmitter. Cholinergic deficits in Alzheimer's disease are typically as- Accordingly, studies have focused on choline acetyltransferase sessed by choline acetyltransferase, the enzyme that synthe- (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), the en- sizes acetylcholine. However, the determining step in acetyl- zyme that synthesizes acetylcholine from choline and acetyl choline formation is choline uptake via a high affinity trans- CoA, and on the high affinity choline transporter that takes up porter in nerve terminal membranes. Evaluating uptake is choline from the synapse and translocates it to the presynaptic difficult because regulatory changes in transporter function terminal. Although nearly all investigations show that Alzhei- decay rapidly postmortem. To overcome this problem, brain mer's disease is characterized by loss of choline acetyltransfer- regions from patients with or without Alzheimer's disease ase (1-3), this enzyme is not the rate-limiting step in acetylcho- were frozen within 4 h of death and examined for both line biosynthesis and its activity is not affected by changes in choline acetyltransferase activity and for binding of [1H]- neuronal activity (4-6); thus, the reduction of choline acetyl- hemicholinium-3 to the choline transporter. Consistent with transferase activity substantiates the loss of cholinergic nerve the loss of cholinergic projections, cerebral cortical areas terminals but gives no indication of the state of cholinergic exhibited marked decreases in enzyme activity whereas the function of the remaining terminals. putamen, a region not involved in Alzheimer's disease, was The issue of cholinergic function in brain regions damaged unaffected. However, [3H]hemicholinium-3 binding was sig- by Alzheimer's disease is vital to understanding both the origins nificantly enhanced in the cortical regions. In the frontal and potential therapy of cognitive impairment. The loss of dopa- cortex, the increase in [3H]hemicholinium-3 binding far ex- minergic function in Parkinson's disease does not lead to overt ceeded the loss of choline acetyltransferase, indicating trans- symptoms until the majority of nerve terminals has degenerated porter overexpression beyond that necessary to offset loss and even then can be offset by administration of the dopamine of synaptic terminals. These results suggest that, in Alzhei- precursor, L-dopa (7); the treatment enables sufficient dopamine mer's disease, the loss of cholinergic function is not dictated synthesis to keep pace with increased impulse activity that off- simply by destruction of nerve terminals, but rather involves sets the deficiency in the number of nerve terminals. In contrast, additional alterations in choline utilization; interventions administering choline, the precursor to acetylcholine, has pro- aimed at increasing the activity of cholinergic neurons may duced variable but largely disappointing results in Alzheimer's thus accelerate neurodegeneration. (J. Clin. Invest. 1994. disease (for review see reference 3), despite the fact that deficits 94:696-702.) Key words: acetylcholine, function in Alzhei- in choline acetyltransferase indicate a smaller synaptic loss than mer's disease * Alzheimer's disease * autopsy, rapid, in Alz- the dopaminergic deficit in Parkinson's disease (8). This dispar- heimer's disease * choline acetyltransferase, in Alzheimer's ity suggests that the cholinergic neurons have a limitation in disease * choline transporter, in Alzheimer's disease their ability to acquire or use choline (1) that transcends the straightforward strategy of simply providing an increase in di- Introduction etary intake of the appropriate neurotransmitter precursor. To an extent, this reflects basic differences in the biosynthetic steps Alzheimer's disease, morphologically characterized by the ab- leading to formation of dopamine as compared with acetylcho- normal presence of senile plaques and neurofibrillary tangles, line. Dopamine is formed from L-dopa by L-aromatic amino acid is distinguished neuroanatomically by the loss of cholinergic decarboxylase, which is in plentiful supply in a wide variety in projections to the cerebral cortex, a deficit that has provided a of cells. Acetylcholine can be formed from choline only focus for studies of potential therapies for cognitive defects (1- cholinergic neurons. Therefore, it is of critical importance that 3). Unfortunately, the rapid postmortem degradation of acetyl- the rate-limiting step in acetylcholine synthesis is the uptake of choline from the synapse and that the activity of the high affinity transporter, located solely on cholinergic nerve terminals, is directly affected by neuronal impulses (4-6). In animal studies, Address correspondence to Dr. T. A. Slotkin, Box 3813/Department of upregulation of choline transport is readily obtained after ma- Pharmacology, Duke University Medical Center, Durham, NC 27710. nipulations that enhance cholinergic tone (4, 6, 9, 10). However, Received for publication 15 February 1994 and in revised form 22 this step has rarely been evaluated in Alzheimer's disease be- March 1994. cause measurement of choline uptake requires the isolation of J. Clin. Invest. nerve terminal particles (synaptosomes) that maintain their via- © The American Society for Clinical Investigation, Inc. bility for only a short period after death; indeed, the component 0021-9738/94/08/0696/07 $2.00 of choline transport specifically associated with increased neu- Volume 94, August 1994, 696-702 ronal impulse activity decays the most rapidly (11). By ob- 696 T. A. Slotkin, C. B. Nemeroff G. Bissette, and F. J. Seidler taining synaptosomes within 2 h postmortem, we have recently Instruments, Inc., Westbury, NY). Depending upon region, the tissue found that choline uptake in cortical regions of patients with homogenate was diluted as much as eightfold before performing choline Alzheimer's disease is not decreased, but rather is elevated (12); acetyltransferase assays, in order to ensure that enzyme activity mea- in keeping with the rapid decay of the impulse-activity-related surements would remain linear over the incubation time used. Assays component of uptake, the elevation is not found when the post- were conducted essentially as described by Lau et al. (15), using 30 of diluted a mortem wait is prolonged (2, 13). These results suggest that, iul homogenate in total volume of 60 p1 containing final concentrations of 60 mM sodium 200 in Alzheimer's disease, the remaining cholinergic nerve termi- phosphate (pH 7.9), mM NaCI, 20 mM choline chloride, 17 mM MgCl2, 1 mM EDTA, 0.2% Triton X- nals are attempting to compensate for synaptic loss by increas- 100, 0.12 mM physostigmine (Sigma Chemical Co., St. Louis, MO), ing their capacity to take up choline, yet, unlike the situation 0.6 mg/ml bovine serum albumin (Sigma Chemical Co.), and 50 p1M for dopaminergic function in Parkinson's disease, they fail to ['4C]acetyl-coenzyme A (DuPont Medical Products, Wilmington, DE; make up for the deficit. sp act 56 mCi/mmol, diluted with unlabeled compound to 6.3 mCi/ A number of uncertainties exist in evaluating the origin mmol). Blanks contained water instead of the tissue homogenate. Sam- and meaning of the changes in high affinity choline uptake in ples were preincubated for 15 min on ice and transferred to a 370C Alzheimer's disease. The uptake process involves not only the water bath for 30 min, and the reaction was terminated by placing the activity of the choline transporter itself, but also the metabolic samples on ice; the labeled acetylcholine was then extracted and counted. The was also status of the terminal (to provide the driving energy for trans- original homogenate analyzed for total protein (16), and the enzyme activity was expressed as micromoles of port), factors influencing the choline concentration gradient (re- acetylcho- line formed per milligram of protein per hour. The daily moval of free cytoplasmic choline its consistency by incorporation into and linearity of the assay was verified by including samples prepared acetylcholine and phospholipids), packaging of acetylcholine in from rat cerebral cortex. synaptic vesicles, and leakage of choline back into the synapse. To assess [3H]hemicholinium-3 binding (DuPont Medical Products; As a first step in delineating the contribution of each of these sp act 121 Ci/mmol), the tissue homogenate was diluted fourfold and factors, the current study evaluates expression of the transporter, sedimented at 40,000 g for 15 min, and the supernatant solution was as evaluated with the specific radioligand, [3H]hemicholinium-3 discarded. The membrane pellet was resuspended