![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Center for Neurodegenerative Disease and Department of Neurology, Emory University School of Medicine, Whitehead Biomedical Research Building, Atlanta, Georgia (L.A.V.-D., A.I.L.); Eppley Institute, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska (A.H., E.G.D., O.L.); and Department of Pharmacology, Vanderbilt University Medical School, Nashville, Tennessee (S.M.F., R.D.B.)
Received July 7, 2003; accepted August 20, 2003
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
2 subunit of nicotinic receptors. M1, M2, and M4 also showed decreased dendritic and cell surface distributions and enhanced intracellular localizations in striatal neurons of AChE-/- mice. mAChR antagonist treatment reversed the shifts in mAChR distribution, indicating that internalized receptors in AChE-/- mice can recover to basal distributions. Finally, AChE-/- mice showed increased sensitivity to mAChR antagonist-induced increases in locomotor activity, demonstrating functional mAChR down-regulation. mAChR downregulation in AChE-/- mice has important implications for the long-term use of cholinesterase inhibitors and other cholinomimetics in treating disorders characterized by perturbed cholinergic function.
; Jerusalinsky et al., 1997; Pisani et al., 2001). Perturbed cholinergic transmission is involved in devastating disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and schizophrenia (Graybiel, 1990; Holt et al., 1999; Pisani et al., 2001). Cholinesterase inhibitors to enhance cholinergic transmission provide the primary treatment for Alzheimer's disease and may ameliorate hyperkinetic movement disorders associated with cholinergic hypofunction such as Huntington's chorea and tardive dyskinesia (Davis and Berger, 1978; Tammenmaa et al., 2002). Furthermore, the antipsychotic properties of these drugs have spurred new interest in the role of cholinomimetics in schizophrenia treatment (White and Cummings, 1996; Shannon et al., 2000) and the neuropsychiatric symptoms of Parkinson's disease (Mori, 2002). Although cholinomimetics show promise in treating a variety of neurological and psychiatric disorders, long-term treatment with cholinesterase inhibitors has been of limited efficacy (Farlow and Evans, 1998; Francis et al., 1999). It is possible that mechanisms that compensate for long-term loss of cholinesterase activity down-regulate the neuronal responsiveness to increased ACh and restrict the effectiveness of these agents. Potential targets for adaptive responses to altered cholinergic transmission include proteins involved in ACh synthesis and release and receptors involved in neurotransmission.
Recently developed AChE knockout (-/-) mice provide a valuable tool for examining the effects of long-term complete and selective abolition of AChE activity (Xie et al., 2000; Duysen et al., 2002). These mice show normal development of cholinergic pathways (Mesulam et al., 2002), but they exhibit tremor, a classic symptom of basal ganglia dysfunction and hypercholinergic activity (Xie et al., 2000). In this study, we investigated the expression and function of key molecules involved in cholinergic transmission in the striatum. The striatum is densely innervated by cholinergic interneurons that are crucial for motor behavior (Pisani et al., 2001), and this structure is highly enriched in cholinergic markers. AChE-/- mice showed dramatic and selective reduction in mAChR protein levels, a marked redistribution of mAChRs to intracellular compartments, up-regulation of the highaffinity choline transporter (CHT), and altered locomotor activity induced by mAChR antagonists. In contrast, there was no change in the activity of choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh, or in the levels of the vesicular ACh transporter (VAChT) and the 2 subunit of nicotinic receptors. These findings indicate that the cholinergic system exhibits a dramatic adaptive response to long-term loss of AChE activity; the most profound effects resulted from down-regulation of mAChR in AChE-/- mice. These results have important therapeutic implications for the design of pharmacotherapeutic agents targeted at long-term modulation of AChE or mAChR activity to treat CNS disorders involving perturbed cholinergic function.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The mice survive to adulthood when maintained on a liquid diet (Duysen et al., 2002).
ChAT Activity. ChAT activity in striatal homogenates was determined as described previously (Levey et al., 1982) except that Ready Organic scintillant (Beckman Coulter Inc., Fullerton CA) was used. Background was subtracted from the values obtained. The assays were performed in triplicate and the data represent an average from three AChE+/+ and three AChE-/- mice.
Immunoblotting and Quantitation. The striatum was dissected and the samples were homogenized in buffer containing 10 mM Tris·Cl, pH 7.4, 1 mM EDTA, pH 8.0, and protease inhibitor cocktail (Roche Diagnostics Corporation, Indianapolis, IN). The samples were solubilized in Laemmli sample buffer, subjected to SDS-polyacrylamide gel electrophoresis and transferred to membrane. After blocking in Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE), blots were incubated in primary antibodies to the following antigens: 2 subunit of the nicotinic ACh receptor (1:200; Covance, Inc., Princeton, NJ), D1 dopamine receptor (1:500; Levey et al., 1993), D2 dopamine receptor (1:100; Levey et al., 1993), M1 (1 µg/ml), M2 (1:500), M4 (1 µg/ml; Levey et al., 1991), VAChT (1 µg/ml; Gilmor et al., 1996), CHT (1 µg/ml; S. Ferguson, A. Levey and R. Blakely, unpublished observations). To control for loading, primary antibody solutions also contained antibodies to either total extracellular signal-regulated kinse 1/2 (1:500; Cell Signaling Technologies, Beverly, MA) or Na+/K+ ATPase (1:40,000; Upstate Biotechnologies, Waltham, MA). Blots were rinsed and incubated in AlexaFluor 680 donkey anti-mouse or anti-rat (1:10,000; Molecular Probes, Eugene, OR) and IRDye 800 goat anti-rabbit (1:10,000; Rockland, Gilbertsville, PA) secondary antibodies in blocking buffer. Blots were scanned, and quantitated with an Odyssey Infrared Imaging System (Li-Cor Biosciences). To quantitate immunoreactivity of mAChRs, a box was drawn around the bands corresponding to M1, M2, and M4, which are approximately 65 kDa. Glycosylation of the receptors alters the molecular mass of the receptors, resulting in a few mAChR-specific bands within the 65-kDa range. The Odyssey program calculated the integrated intensity of the bands and subtracted background from the values. Background, or 0% immunoreactivity, is calculated using pixels around the perimeter of the area being quantified. The mAChR immunoreactivity for each mouse (three individual AChE+/+ mice and three AChE-/- mice) was normalized to the loading control within each lane. The average mAChR immunoreactivity (± S.E.M.) in the AChE-/- mice was expressed as a percentage of the average immunoreactivity (± S.E.M.) in the AChE+/+ mice. Similar methods were performed for quantitation of other markers. Immunoblotting was performed a minimum of three times and data were analyzed with SPSS software (SPSS Inc., Chicago, IL) using analysis of variance with Fisher's least significant difference post hoc test or independent t test.
Immunofluorescence and Quantitation of Confocal Images. Immunofluorescence, confocal imaging, and quantitation were performed as described previously (Zhang et al., 2002a). Coronal sections were incubated in antibodies to the following antigens diluted in buffer containing 50 µg/ml biotin and 1% normal horse serum: D1 (1:200), D2 (1 µg/ml), M1 (1 µg/ml), M2 (1:100), M4 (1 µg/ml), substance P (neurokinin 1) receptor (1:500; Chemicon International, Temecula, CA). Some sections were also treated with Hoechst 33258 (1:10,000). Unless otherwise indicated in the figure legends, all immunostaining was performed in a minimum of three AChE+/+ mice and three AChE-/- mice.
Drug Treatments. Mice received three intraperitoneal injections of saline or 5 mg/kg atropine dissolved in saline every 30 min; the total time of treatment was 90 min. The mice were then perfused according to the methods described above.
Locomotor Activity. Motor behavior was tested using a motion detector consisting of a dark box in a sound-proof room containing the home cage and a red light-emitting diode. Horizontal activity was measured as the number of photobeam interruptions. Mice were acclimated to the test cage for 2 h before the experiment was begun. Each animal received s.c. injections of 0.05 mg/kg or 0.5 mg/kg scopolamine (Sigma, St. Louis, MO) dissolved in 0.1 M potassium phosphate, pH 7.0, or injections of 0.1 M potassium phosphate alone (vehicle). Locomotor activity was measured for 90 min in consecutive 2-min intervals.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Jope, 1979) and changes in CHT may mediate adaptive response to altered synaptic metabolism of ACh. CHT expression was significantly increased by 
60% in striatal homogenates from AChE-/- mice relative to AChE+/+ control mice (Fig. 1c).
|
Cholinergic Expression in the Brains of AChE-/- Mice. Changes in cholinergic receptor expression could modulate neuronal responsiveness to increased stimulation in response to chronically reduced synaptic ACh metabolism. Because the striatum contains three distinct mAChR proteins (Levey et al., 1991), M1, M2, and M4, these subtypes were measured individually. Levels of all three mAChRs were substantially reduced in homogenates from AChE-/- striatum compared with AChE+/+ controls (Fig. 2, a-c). Compared with wild-type mice, quantitation by immunoblot of receptor levels in AChE-/- mice showed a statistically significant reduction of M1, M2, and M4 immunoreactivity in the striatum by 40, 56, and 64%, respectively. Therefore, the M1, M2, and M4 mAChRs showed consistent and dramatic reductions in the striatum of AChE-/- mice. Nicotinic cholinergic receptors expressed in the striatum also provide a potential target for adaptive responses to reduced ACh metabolism. The 2 subunit of the nicotinic receptor, which is expressed in striatal dopamine terminals (Zoli et al., 2002), showed similar levels in AChE-/- mice relative to AChE+/+ mice (Fig. 2f). Hence, modulatory G protein-coupled mAChR, but not inotropic nicotinic receptors, are altered in AChE-/- mice.
|
Expression of Receptors Involved in Dopaminergic Transmission in the Striatum of AChE-/- Mice. A balance between the cholinergic and dopaminergic system in the striatum is important for motor behavior (Graybiel, 1990), and M1 and M4 play important roles in striatal dopaminergic transmission (Gomeza et al., 1999b; Gerber et al., 2001; Zhang et al., 2002b). M1 and M4 in medium spiny neurons are coexpressed with the D1 and D2 dopamine receptors, which are also members of the G protein-coupled receptor family (Graybiel, 1990; Ince et al., 1997; Yan et al., 2001). Given the marked changes in expression of mAChRs, we examined the effect of diminished ACh metabolism in AChE-/- mice on expression of dopamine receptors. Levels of D1 and D2 dopamine receptor immunoreactivities in the striatum were not significantly altered in AChE-/- mice relative to AChE+/+ control mice (Fig. 2, d and e). Overall, these data demonstrate that diminished ACh metabolism in the striatum selectively reduced mAChR protein levels without affecting dopamine receptors.
Localization of Postsynaptic mAChRs to Dendrites and the Cell Surface in AChE-/- Mice. Agonist-induced mAChR endocytosis promotes down-regulation (Tsuga et al., 1998; Shockley et al., 1999); thus, increased internalization may contribute to the reduced mAChR levels in AChE-/- mice. In addition, decreased availability of mAChRs at the cell surface and dendrites may provide another mechanism by which postsynaptic neurons respond to long-term diminished synaptic metabolism of ACh. The subcellular distributions of the postsynaptically expressed M1 and M4 mAChRs were analyzed in medium spiny neurons of AChE+/+ and AChE-/- mice (Fig. 3, a and b). Previous immunohistochemical and electron microscopic studies demonstrate localization of M1 and M4 to cell bodies and dendrites of medium spiny neurons of the striatum (Hersch et al., 1994). In the striatum of AChE+/+ mice, M1 and M4 showed a dendritic neuropil distribution (Fig. 3, +/+). In AChE-/- mice, both M1 (Fig. 3a) and M4 (Fig. 3b) showed markedly reduced staining intensity in the neuropil. In addition, the appearance of prominent staining around the nuclei of some neurons in AChE-/- mice indicates increased intracellular localization of the receptors (Fig. 3, -/-).
|
The substance P receptor (SPR) and the M2 mAChR are both expressed in cholinergic interneurons of the striatum (Kaneko et al., 1993). Double labeling for M2 and SPR was used to evaluate the redistribution of mAChRs in neurons of AChE-/- mice. In striatal interneurons of AChE+/+ mice, M2 localized predominantly to the perikaryal cell surface (Fig. 3c) with occasional regions of enhanced immunoreactivity in subdomains at the cell surface and scattered intracellular puncta. M2 also localized to small puncta throughout the striatum, consistent with localization to presynaptic terminals (Hersch et al., 1994). The SPR showed a continuous distribution along the plasma membrane and colocalized with M2 within domains along the cell surface (Fig. 3, arrows). In AChE-/- mice, the distribution of the SPR was not altered, but M2 no longer localized to the cell surface and showed an enhanced intracellular distribution (Fig. 3c, bottom). These data confirm the dramatic redistribution of mAChRs from the cell surface to intracellular compartments in AChE-/- mice. Furthermore, the shifts in subcellular distribution are selective for mAChRs and do not reflect generalized alterations or impairment in G protein-coupled receptor trafficking.
Altered M4 Subcellular Localization Results from Enhanced Activity-Dependent Internalization in D1-Dopamine Receptor Expressing Neurons. mRNA studies demonstrate that M4 is expressed in both D1-expressing striatonigral medium spiny neurons and D2-expressing striatopallidal medium spiny neurons (Yan et al., 2001). D2 is enriched in the striatal matrix in which AChE is also enriched, whereas D1 is more concentrated in AChE-poor striosomes of the striatum (Graybiel, 1990). In AChE+/+ mice, M4 mAChR protein and D2 dopamine receptor protein do not colocalize in the striatum (Fig. 4a). However, M4 and D1 colocalize extensively in the dendritic neuropil (Fig. 4b), consistent with quantitative electron microscopic studies, demonstrating that approximately 95% of M4 protein colocalizes with D1 protein-expressing medium spiny neurons, whereas it colocalizes with only 1% of D2 protein-expressing neurons (Ince et al., 1997). Therefore, the redistribution of M4 in AChE-/- mice occurs selectively in D1-expressing striatonigral medium spiny neurons.
|
The M4 redistribution in AChE-/- mice relative to the AChE+/+ control mice could result from ACh-induced internalization of the receptors or impaired trafficking of the receptors from biosynthetic compartments to the cell surface. To distinguish among these possibilities, the mice were treated with the mAChR antagonist atropine to prevent ACh-induced mAChR activation and internalization. AChE+/+ mice and AChE-/- mice received injections of atropine or saline (total time of treatment was 90 min). In saline-treated AChE-/- mice, M4 showed reduced localization to dendritic neuropil and enhanced intracellular localization within cell bodies (Fig. 4b). D1 distribution did not seem to be altered in AChE-/- mice. M4 and D1 showed minimal colocalization in AChE-/- mice as a result of the altered subcellular distribution of M4. After a 90-min treatment with atropine, M4 in AChE-/- mice showed a neuropil distribution similar to that of saline-treated AChE+/+ mice, and M4 and D1 colocalized extensively.
Quantification of M4 and D1 colocalization in the striatum showed that M4 overlap with D1 was significantly reduced by 52% in saline-treated AChE-/- mice compared with saline-treated AChE+/+ mice (Fig. 4c). Treatment with atropine significantly increased M4 /D1 colocalization in the AChE-/- mice to levels equivalent to those of AChE+/+ mice. Atropine-induced recovery of M4 to basal distributions may result from increased protein expression of M4 or redistribution of M4 from intracellular compartments to the cell surface and dendritic neuropil. Immunoblots of M4 were thus performed after 90-min atropine treatments to evaluate possible recovery of protein levels (Fig. 4d). M4 immunoreactivity in atropine-treated AChE-/- mice did not increase relative to saline-treated AChE-/- mice. Therefore, inhibiting acetylcholine-induced M4 activation for 90 min did not increase M4 protein levels but allowed M4 to traffic from intracellular compartments back to the dendritic neuropil. Similarly, M2 also returned to the cell surface of cholinergic interneurons after atropine treatment without an increase in protein levels (data not shown). Overall, these data show that although mAChR localization is dramatically altered in AChE-/- mice, the receptors can rapidly return to a localization similar to control mice, and the redistribution of mAChRs to intracellular compartments in AChE-/- mice results from acetylcholine-induced internalization.
Effects of mAChR Blockade on Locomotor Activity in AChE-/- Mice. To determine whether the altered expression and distribution of striatal mAChRs are reflected in behavioral changes, we examined locomotor activity in the AChE-/- mice. Administration of mAChR antagonists, such as the high-affinity antagonist scopolamine, increases locomotor activity (Sipos et al., 1999), and M1 -/- mice and M4 -/- mice show enhanced locomotor activity (Gomeza et al., 1999b; Gerber et al., 2001; Miyakawa et al., 2001). Therefore, the changes M1 and M4 in AChE-/- mice may induce similar behavioral changes or cause enhanced sensitivity to the effects of mAChR antagonists on locomotor activity. At baseline, AChE+/+ mice and AChE-/- mice did not show a statistically significant difference in locomotor activity (Fig. 5). After administration of a low dose (0.05 mg/kg) of scopolamine, AChE-/- mice showed a dramatic increase in locomotor activity compared with injection of vehicle. The AChE+/+ mice did not show increased locomotor activity at this dose, and the difference in locomotor activity between the AChE+/+ mice and AChE-/- mice was statistically significant. Administration of 0.5 mg/kg scopolamine increased locomotor activity in both AChE+/+ mice and AChE-/- mice, and the two genotypes did not show a statistically significant difference in motor activity after administration of the higher dose of scopolamine. Therefore, AChE-/- mice showed markedly enhanced sensitivity to mAChR antagonist-induced increases in motor behavior.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
2 subunit of nicotinic ACh receptors. In addition, M1, M2, and M4 show dramatic shifts in cell surface and dendritic localizations in AChE-/- mice that are atropine-reversible. The down-regulation and altered distribution of mAChRs results in altered motor function in the AChE-/- mice, with enhanced sensitivity to mAChR antagonist-induced increases in locomotor activity. This is the first study to demonstrate that the CNS responds to complete and selective loss of AChE activity by (1) selective regulation of CHT, which mediates the rate-limiting step in ACh synthesis, and (2) selectively decreasing expression of all mAChR subtypes expressed in the striatum without affecting nicotinic ionotropic acetylcholine receptors or GPCRs activated by dopamine. These findings have important implications for understanding the regulation of mAChRs in the CNS and the application of AChE inhibitors and mAChR agonists as therapeutic agents.
High-Affinity Choline Transporter Protein Levels Are Increased in Response to Diminished Metabolism of ACh. High-affinity choline uptake is the rate-limiting step in the synthesis of ACh. Similar to AChE-/- mice, mice in which the dopamine transporter gene has been deleted (dopamine transporter -/- mice) show hyperdopaminergic activity (Giros et al., 1996). These mice show reduced expression of tyrosine hydroxylase, the rate-limiting enzyme involved in dopamine synthesis, and the vesicular monoamine transporter, which mediates uptake of dopamine into synaptic vesicles (Jaber et al., 1999). In contrast to the findings in DAT-/- mice, ChAT activity and VAChT expression were not affected in AChE-/- mice. However, AChE-/- mice did show a dramatic increase in CHT expression. Regulation of signaling cascades through presynaptic mAChR activation may be involved in controlling CHT expression, possibly through modulation of cAMP activity (Breer and Knipper, 1990). In addition to synthesis of ACh, presynaptic choline is used for synthesis of phosphatidylcholine, a component of the plasma membrane. Therefore, CHT may be increased as a response to reduced metabolism of ACh into choline and acetic acid in AChE-/- mice. mAChRs Show Selective Reductions in Protein Levels and Shifts in Subcellular Distribution in AChE-/- mice. The most dramatic response found in AChE-/- mice was decreased immunoreactivity of striatal M1, M2, and M4. Levels of the D1 and D2 dopamine receptors were not altered in AChE-/- mice, demonstrating the selectivity of the effect for cholinergic receptors. Furthermore, levels of the 2 subunit of the nicotinic receptor was not altered in AChE-/- mice, suggesting that metabotropic mAChRs, but not nicotinic ion channels, are particularly sensitive to persistently diminished ACh metabolism. Northern blot analyses demonstrate that mRNA levels of M1, M2, and M4 are not altered in AChE-/- mice (Li et al., 2003). Therefore, reduced mAChR immunoreactivity most probably results from receptor degradation and not from decreased mAChR synthesis, consistent with electron microscopy studies showing that after long-term stimulation, a proportion of mAChRs localize to intracellular organelles associated with degradation (Liste et al., 2002).
M1, M2, and M4 also show decreased localization to the cell surface and dendrites and an enhanced intracellular distribution in AChE-/- mice. These data support previous findings that the M2 mAChR shows decreased cell surface localization in AChE-/- mice (Bernard et al., 2003; Decossas et al., 2003) and extend these findings to all the mAChR sub-types predominately expressed in the CNS. Treatment of AChE-/- mice with atropine for 90 min causes mAChR subcellular localization to recover to distributions similar to those of AChE+/+ mice, confirming that the shift in mAChR distribution results from ACh-induced internalization, not from impaired trafficking from biosynthetic pathways to the cell surface. The recovery of mAChRs to basal distributions in atropine-treated AChE-/- mice results from a return of intracellular receptors back to the cell surface, not from increased receptor synthesis, demonstrating that internalized mAChRs in neurons can rapidly recycle to the cell surface. Overall, these data indicate that mAChR internalization after long-term stimulation and consequently decreased mAChR localization to dendrites and the cell surface provides a compensatory mechanism by which the CNS responds to persistently enhanced cholinergic stimulation.
mAChR Function Is Impaired in AChE-/- Mice. Decreased expression and receptor redistribution corresponds to altered mAChR function in AChE-/- mice, as demonstrated by enhanced sensitivity to scopolamine-induced increases in locomotor behavior. The increased sensitivity to antagonist probably results from mAChR down-regulation in the striatum. M1 and M4 play important roles in the control of locomotor activity, as demonstrated by increased locomotor activity in M1-/- mice and M4-/- mice (Gomeza et al., 1999b; Gerber et al., 2001; Miyakawa et al., 2001). It has been suggested that M1 and M4 stimulation decreases motor behavior by altering activity of the striatopallidal and striatonigral pathways (Gomeza et al., 1999b; Miyakawa et al., 2001). Reduction or inhibition of M1 and M4 would consequently increase locomotor activity. Low doses of scopolamine increased motor behavior in AChE-/- mice but not in AChE+/+ mice. Because mAChR levels are reduced in AChE-/- mice, lower concentrations of antagonist are required to produce increased locomotor activity. Thus, AChE-/- mice showed enhanced sensitivity to mAChR antagonist-induced increases in motor activity, indicative of functional down-regulation of striatal mAChRs.
Therapeutic Implications. The profound alterations in mAChR expression and subcellular localization after long-term loss of AChE activity in AChE-/- mice have important therapeutic implications for cholinergic drug therapy in humans. Long-term treatment with cholinesterase inhibitors is the primary therapy for Alzheimer's disease (Farlow and Evans, 1998; Francis et al., 1999) and shows promise for other disorders, including dementia associated with Parkinson's disease, Huntington's chorea, and tardive dyskinesia (Davis and Berger, 1978; Mori, 2002; Tammenmaa et al., 2002). Antipsychotic effects of AChE inhibitors have also fostered development of cholinomimetic treatments for schizophrenia (White and Cummings, 1996; Shannon et al., 2000). Because mAChR stimulation by enhanced synaptic activity of ACh most probably mediates the therapeutic response in these conditions, the marked down-regulation of mAChRs after long-term inhibition of AChE activity would limit efficacy of cholinomimetic drug treatment for CNS disorders.
The implications of the findings in this study may also extend beyond mAChRs in the CNS. For example, increased acetylcholine resulting from acetylcholinesterase inhibition will persistently activate mAChRs in the neuromuscular junction, smooth muscles of airway, the heart, exocrine glands, and gastrointestinal and urinary tracts. Therefore, expression and subcellular localization of mAChRs are most probably altered throughout the body. In addition, similar to mAChR activation by cholinomimetics, many clinical treatments persistently stimulate various GPCRs, such as prostaglandins for vascular disease, serotonergic receptors for depression, 
-adrenergic receptors for hypertension, and hypothalamic hormones used to treat a wide variety of disorders, ranging from tumors to infertility to diabetes. In fact, dopamine receptors of Parkinson's disease patients show an increased intracellular distribution after prolonged treatment with levodopa (Muriel et al., 1999). Thus, similar to mAChRs, long-term administration of a variety of therapeutics may alter subcellular localization and function of multiple GPCR subtypes.
Although GPCRs may show dramatically altered expression and subcellular localization after long-term drug administration, it may be possible to improve the efficacy of therapies that stimulate GPCRs. For example, we show that the effects on mAChR subcellular localization are rapidly reversible in the AChE-/- mice. Therefore, it is possible that other variables, such as the temporal pattern of cholinesterase inhibitor administration or the degree of inhibition, can influence the internalization, recycling, and down-regulation of mAChRs. In addition, the efficacy of cholinomimetic therapies may be enhanced by the development of selective mAChR ligands that activate the receptors without causing internalization or extensive down-regulation, as has been demonstrated for nonselective mAChR partial agonists (Nathanson et al., 1984) and µ-opioid receptor ligands (Keith et al., 1998). Hence, our findings in AChE-deficient mice identify novel mechanisms that regulate cholinergic neurotransmission, providing potential insights to the therapeutic limitations of long-term inhibition of AChE activity in humans. A better understanding of the factors regulating expression and localization of molecules involved in cholinergic transmission is important to improve the efficacy of long-term cholinergic drug treatments.
|
Footnotes |
|---|
Portions of this work have been previously presented as part of the doctoral dissertation of L.A.V.-D.
ABBREVIATIONS: ACh, acetylcholine; AChE, acetylcholinesterase; mAChR, muscarinic receptor; CHT, high affinity choline transporter; ChAT, choline acetyltransferase; VAChT, vesicular acetylcholine transporter; SPR, substance P receptor; CNS, central nervous system; GPCR, G protein-coupled receptor.
Address correspondence to: Dr. A. I. Levey, Emory University School of Medicine, Whitehead Biomedical Research Building, 615 Michael St., 5th Floor, Atlanta, GA 30322. E-mail: alevey{at}emory.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Breer H and Knipper M (1990) Regulation of high affinity choline uptake. J Neurobiol 21: 269-275.[CrossRef][Medline]
Davis KL and Berger PA (1978) Pharmacological investigations of the cholinergic imbalance hypotheses of movement disorders and psychosis. Biol Psychiatry 13: 23-49.[Medline]
Decossas M, Bloch B, and Bernard V (2003) Trafficking of the muscarinic m2 autoreceptor in cholinergic basalocortical neurons in vivo: differential regulation of plasma membrane receptor availability and intraneuronal localization in acetylcholinesterase-deficient and -inhibited mice. J Comp Neurol 462: 302-314.[CrossRef][Medline]
Duysen EG, Stribley JA, Fry DL, Hinrichs SH, and Lockridge O (2002) Rescue of the acetylcholinesterase knockout mouse by feeding a liquid diet; phenotype of the adult acetylcholinesterase deficient mouse. Brain Res Dev Brain Res 137: 43-54.[CrossRef][Medline]
Farlow MR and Evans RM (1998) Pharmacologic treatment of cognition in Alzheimer's dementia. Neurology 51: S36-S44; discussion S65-S67.
Francis PT, Palmer AM, Snape M, and Wilcock GK (1999) The cholinergic hypothesis of Alzheimer's disease: a review of progress. J Neurol Neurosurg Psychiatry 66: 137-147.
Gerber DJ, Sotnikova TD, Gainetdinov RR, Huang SY, Caron MG, and Tonegawa S (2001) Hyperactivity, elevated dopaminergic transmission and response to amphetamine in M1 muscarinic acetylcholine receptor-deficient mice. Proc Natl Acad Sci USA 98: 15312-15317.
Gilmor ML, Nash NR, Roghani A, Edwards RH, Yi H, Hersch SM, and Levey AI (1996) Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles. J Neurosci 16: 2179-2190.
Giros B, Jaber M, Jones SR, Wightman RM, and Caron MG (1996) Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature (Lond) 379: 606-612.[CrossRef][Medline]
Gomeza J, Shannon H, Kostenis E, Felder C, Zhang L, Brodkin J, Grinberg A, Sheng H, and Wess J (1999a) Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 96: 1692-1697.
Gomeza J, Zhang L, Kostenis E, Felder C, Bymaster F, Brodkin J, Shannon H, Xia B, Deng C, and Wess J (1999b) Enhancement of D1 dopamine receptor-mediated locomotor stimulation in M(4) muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 96: 10483-10488.
Graybiel AM (1990) Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci 13: 244-254.[CrossRef][Medline]
Hersch SM, Gutekunst CA, Rees HD, Heilman CJ, and Levey AI (1994) Distribution of m1-m4 muscarinic receptor proteins in the rat striatum: light and electron microscopic immunocytochemistry using subtype-specific antibodies. J Neurosci 14: 3351-3363.[Abstract]
Holt DJ, Herman MM, Hyde TM, Kleinman JE, Sinton CM, German DC, Hersh LB, Graybiel AM, and Saper CB (1999) Evidence for a deficit in cholinergic interneurons in the striatum in schizophrenia. Neuroscience 94: 21-31.[CrossRef][Medline]
Ince E, Ciliax BJ, and Levey AI (1997) Differential expression of D1 and D2 dopamine and m4 muscarinic acetylcholine receptor proteins in identified striatonigral neurons. Synapse 27: 357-366.[CrossRef][Medline]
Jaber M, Dumartin B, Sagne C, Haycock JW, Roubert C, Giros B, Bloch B, and Caron MG (1999) Differential regulation of tyrosine hydroxylase in the basal ganglia of mice lacking the dopamine transporter. Eur J Neurosci 11: 3499-3511.[CrossRef][Medline]
Jerusalinsky D, Kornisiuk E, and Izquierdo I (1997) Cholinergic neurotransmission and synaptic plasticity concerning memory processing. Neurochem Res 22: 507-515.[CrossRef][Medline]
Jope RS (1979) High affinity choline transport and acetylCoA production in brain and their roles in the regulation of acetylcholine synthesis. Brain Res 180: 313-344.[CrossRef][Medline]
Kaneko T, Shigemoto R, Nakanishi S, and Mizuno N (1993) Substance P receptor-immunoreactive neurons in the rat neostriatum are segregated into somatostatinergic and cholinergic aspiny neurons. Brain Res 631: 297-303.[CrossRef][Medline]
Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Lissin DV, Monteillet-Agius G, Stewart PL, Evans CJ, and von Zastrow M (1998) mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain. Mol Pharmacol 53: 377-384.
Kuhar MJ and Murrin LC (1978) Sodium-dependent, high affinity choline uptake. J Neurochem 30: 15-21.[Medline]
Levey AI, Hersch SM, Rye DB, Sunahara RK, Niznik HB, Kitt CA, Price DL, Maggio R, Brann MR, Ciliax BJ, et al. (1993) Localization of D1 and D2 dopamine receptors in brain with subtype-specific antibodies. Proc Natl Acad Sci USA 90: 8861-8865.
Levey AI, Kitt CA, Simonds WF, Price DL, and Brann MR (1991) Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies. J Neurosci 11: 3218-3226.[Abstract]
Levey AI, Rye DB, and Wainer BH (1982) Immunochemical studies of bovine and human choline-O-acetyltransferase using monoclonal antibodies. J Neurochem 39: 1652-1659.[CrossRef][Medline]
Li B, Duysen EG, Volpicelli LA, Levey AI, and Lockridge O (2003) Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice. Pharmacol Biochem Behav 74: 977-986.[CrossRef][Medline]
Liste I, Bernard V, and Bloch B (2002) Acute and chronic acetylcholinesterase inhibition regulates in vivo the localization and abundance of muscarinic receptors m2 and m4 at the cell surface and in the cytoplasm of striatal neurons. Mol Cell Neurosci 20: 244-256.[CrossRef][Medline]
Mesulam MM, Guillozet A, Shaw P, Levey A, Duysen EG, and Lockridge O (2002) Acetylcholinesterase knockouts establish central cholinergic pathways and can use butyrylcholinesterase to hydrolyze acetylcholine. Neuroscience 110: 627-639.[CrossRef][Medline]
Miyakawa T, Yamada M, Duttaroy A, and Wess J (2001) Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor. J Neurosci 21: 5239-5250.
Mori S (2002) Responses to donepezil in Alzheimer's disease and Parkinson's disease. Ann N Y Acad Sci 977: 493-500.[Medline]
Muriel MP, Bernard V, Levey AI, Laribi O, Abrous DN, Agid Y, Bloch B, and Hirsch EC (1999) Levodopa induces a cytoplasmic localization of D1 dopamine receptors in striatal neurons in Parkinson's disease. Ann Neurol 46: 103-111.[CrossRef][Medline]
Nathanson NM, Holttum J, Hunter DD, and Halvorsen SW (1984) Partial agonist activity of oxotremorine at muscarinic acetylcholine receptors in the embryonic chick heart. J Pharmacol Exp Ther 229: 455-458.
Pisani A, Bonsi P, Picconi B, Tolu M, Giacomini P, and Scarnati E (2001) Role of tonically-active neurons in the control of striatal function: cellular mechanisms and behavioral correlates. Prog Neuropsychopharmacol Biol Psychiatry 25: 211-230.[CrossRef][Medline]
Shannon HE, Rasmussen K, Bymaster FP, Hart JC, Peters SC, Swedberg MD, Jeppesen L, Sheardown MJ, Sauerberg P and Fink-Jensen A (2000) Xanomeline, an M(1)/M(4) preferring muscarinic cholinergic receptor agonist, produces antipsychotic-like activity in rats and mice. Schizophr Res 42: 249-259.[CrossRef][Medline]
Shockley MS, Tolbert LM, Tobin AB, Nahorski SR, Sadee W, and Lameh J (1999) Differential regulation of muscarinic M1 and M3 receptors by a putative phosphorylation domain. Eur J Pharmacol 377: 137-146.[CrossRef][Medline]
Sipos ML, Burchnell V, and Galbicka G (1999) Dose-response curves and time-course effects of selected anticholinergics on locomotor activity in rats. Psychopharmacology (Berl) 147: 250-256.[CrossRef][Medline]
Tammenmaa IA, McGrath JJ, Sailas E, and Soares-Weiser K (2002) Cholinergic medication for neuroleptic-induced tardive dyskinesia. Cochrane Database Syst Rev (3): CD000207 [GenBank] .
Tsuga H, Kameyama K, Haga T, Honma T, Lameh J, and Sadee W (1998) Internalization and down-regulation of human muscarinic acetylcholine receptor m2 sub-types. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2. J Biol Chem 273: 5323-5330.
White KE and Cummings JL (1996) Schizophrenia and Alzheimer's disease: clinical and pathophysiologic analogies. Compr Psychiatry 37: 188-195.[CrossRef][Medline]
Xie W, Stribley JA, Chatonnet A, Wilder PJ, Rizzino A, McComb RD, Taylor P, Hinrichs SH, and Lockridge O (2000) Postnatal developmental delay and super-sensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase. J Pharmacol Exp Ther 293: 896-902.
Yan Z, Flores-Hernandez J, and Surmeier DJ (2001) Coordinated expression of muscarinic receptor messenger RNAs in striatal medium spiny neurons. Neuroscience 103: 1017-1024.[CrossRef][Medline]
Zhang W, Basile AS, Gomeza J, Volpicelli LA, Levey AI, and Wess J (2002a) Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice. J Neurosci 22: 1709-1717.
Zhang W, Yamada M, Gomeza J, Basile AS, and Wess J (2002b) Multiple muscarinic acetylcholine receptor subtypes modulate striatal dopamine release, as studied with M1-M5 muscarinic receptor knock-out mice. J Neurosci 22: 6347-6352.
Zoli M, Moretti M, Zanardi A, McIntosh JM, Clementi F, and Gotti C (2002) Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum. J Neurosci 22: 8785-8789.
This article has been cited by other articles:
|
|
 |
|
  B. Li, E. G. Duysen, M. Carlson, and O. Lockridge The Butyrylcholinesterase Knockout Mouse as a Model for Human Butyrylcholinesterase Deficiency J. Pharmacol. Exp. Ther., March 1, 2008; 324(3): 1146 - 1154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Simpson, D. Beavis, A. Leddy, S. Ball, and I. Johnson Naturalistic audit of NICE criteria for the use of cholinesterase inhibitors Psychiatr. Bull., November 1, 2005; 29(11): 410 - 412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Christie and S. M. Hersch Demonstration of nondeclarative sequence learning in mice: Development of an animal analog of the human serial reaction time task Learn. Mem., November 1, 2004; 11(6): 720 - 723. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Ferguson and R. D. Blakely The Choline Transporter Resurfaces: New Roles for Synaptic Vesicles? Mol. Interv., February 1, 2004; 4(1): 22 - 37. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
