Cytosolic Choline Research Articles

Choline in Pediatric Depression

The prefrontal cortex has been previously implicated in the neuropathology of major depressive disorder (MDD). Hence, we used proton magnetic resonance spectroscopy (1H-MRS) to examine choline levels in the prefrontal cortex of youth with major depression. Twelve age- and sex-matched case-control pairs were examined (age range 10 to 18 years, 7 females and 5 males in each group). All subjects were treatment naive at the time of the scan. A long echo 1H-MRS scan was acquired from the right prefrontal cortex (4cc) in all subjects. Right prefrontal Choline/Creatine ratios were elevated in the youth with mood disorders (F1, 11=10.741, p=0.007) as compared with healthy controls. These findings suggest that prefrontal cytosolic choline may be increased in youth with MDD in comparison with healthy controls. This is consistent with reported findings in both adults and adolescents and suggests that MDD in youth is associated with alterations in choline metabolism in the prefrontal cortex.

Cytosolic choline acetyltransferase binds specifically to cholinergic plasma membrane of rat brain synaptosomes to generate membrane-bound enzyme.

Uncovering the way membrane-bound choline acetyltransferase (ChAT) interacts with membranes and with which membrane in cholinergic neurons may help in understanding its role in acetylcholine metabolism. Subfractionation of rat hippocampal synaptosomes aiming to separate synaptic vesicles from plasma membranes shows that membrane-bound ChAT is bound to plasma membrane. Either detergents or urea and alkali can solubilize membrane-bound enzyme. Detergent-solubilized enzyme has a higher sedimentation rate than urea-alkali solubilized or cytosolic ChAT. Once dissociated, membrane-bound ChAT reassociates specifically with cholinergic plasma membranes, a process that was abolished by previous treatment of membranes with trypsin. Cytosolic ChAT behaves similarly. Thus, in cholinergic synaptosomes, ChAT exists as cytosolic and peripheral activity. Cytosolic ChAT generates peripheral enzyme most probably by interacting with a protein of plasma membrane of cholinergic nerve terminals. This "receptor" protein might regulate the amount of membrane-bound ChAT in cholinergic neurons.

Hydrogen proton magnetic resonance spectroscopy in autism: preliminary evidence of elevated choline/creatine ratio.

Hydrogen proton magnetic resonance spectroscopy is only beginning to be studied in autistic individuals. We report an association between hydrogen proton magnetic resonance spectroscopy choline/creatine ratios and severity of autism as measured by the Children's Autistic Rating Scale (Pearson r = .657, P = .04) in 10 autistic children. Hydrogen proton magnetic resonance spectroscopy choline/creatine ratio measures the concentration of cytosolic choline including free choline used in the synthesis of acetylcholine. Elevation in this ratio has been interpreted as a result of membrane degradation such as caused by a tumor or, alternatively, as a result of choline synthesis associated with increased cellular proliferation. Recent neuropathologic evidence has implicated disruption of acetylcholine transmission in the brains of autistic adults. A case-controlled study of hydrogen proton magnetic resonance spectroscopy choline/creatine ratios is warranted.

Brain Proton Magnetic Resonance Spectroscopy in Alzheimer Disease: Changes After Treatment With Xanomeline

Patients with mild-to-moderate Alzheimer disease received transdermal xanomeline, an M1-selective cholinergic agonist, or placebo for 4 months. Clinical assessments and proton magnetic resonance spectroscopic imaging examinations were carried out at baseline, and after 8 and 16 weeks of treatment. There was a positive correlation between change from baseline in parietal lobe gray-matter cytosolic choline, expressed in terms of choline/creatine resonance ratios, and cognitive performance as measured with the Alzheimer's Disease Assessment Scale Cognitive Subscale. Specifically, increased levels of cytosolic choline, a precursor pool for acetylcholine synthesis, were associated with greater progression in memory impairment during treatment. Patients with mild-to-moderate Alzheimer disease received transdermal xanomeline, an M1-selective cholinergic agonist, or placebo for 4 months. Clinical assessments and proton magnetic resonance spectroscopic imaging examinations were carried out at baseline, and after 8 and 16 weeks of treatment. There was a positive correlation between change from baseline in parietal lobe gray-matter cytosolic choline, expressed in terms of choline/creatine resonance ratios, and cognitive performance as measured with the Alzheimer's Disease Assessment Scale Cognitive Subscale. Specifically, increased levels of cytosolic choline, a precursor pool for acetylcholine synthesis, were associated with greater progression in memory impairment during treatment.

Brain Proton Magnetic Resonance Spectroscopy in Alzheimer Disease: Changes After Treatment With Xanomeline

Patients with mild-to-moderate Alzheimer disease received transdermal xanomeline, an M1-selective cholinergic agonist, or placebo for 4 months. Clinical assessments and proton magnetic resonance spectroscopic imaging examinations were carried out at baseline, and after 8 and 16 weeks of treatment. There was a positive correlation between change from baseline in parietal lobe gray-matter cytosolic choline, expressed in terms of choline/creatine resonance ratios, and cognitive performance as measured with the Alzheimer's Disease Assessment Scale Cognitive Subscale. Specifically, increased levels of cytosolic choline, a precursor pool for acetylcholine synthesis, were associated with greater progression in memory impairment during treatment.

Left medial temporal cytosolic choline in early onset depression.

Previous studies have linked the choline (Cho) resonance seen in proton magnetic resonance spectroscopy (1H-MRS) to major depressive disorder (MDD). We endeavoured to clarify the possible involvement of cytosolic choline in the amygdala (anterior medial temporal region) of juvenile subjects with MDD. A total of 11 age- and sex-matched MDD and control pairs aged 14 to 18 years participated in long-echo proton magnetic resonance spectroscopic imaging (1H-MRSI) of the amygdala. Compounds available include N-acetyl-aspartate (NAA), creatine-phosphocreatine (Cr) and choline-containing compounds. Subjects with depression demonstrated lower left amygdala Cho-Cr ratios, compared with control subjects (paired t = 2.624, df 10, P = 0.025). Left amygdala NAA-Cr and right amygdala Cho-Cr and NAA-Cr did not differ significantly between subjects with depression and control subjects. In subjects with depression, simple regression revealed a negative trend between left amygdala Cho-Cr and Beck Depression Inventory (BDI) score (F = 3.509, P = 0.098). Right amygdala NAA-Cr and Cho-Cr did not differ significantly. Cytosolic choline appears to be involved in the pathophysiology of early-onset MDD, likely secondary to corticosteroid-neuroendocrine-driven changes.

Increased medial thalamic choline in pediatric obsessive-compulsive disorder as detected by quantitative in vivo spectroscopic imaging.

The thalamus has been implicated in the pathophysiology of obsessive-compulsive disorder. Using a multislice spectroscopic imaging sequence, we reported reductions in right and left medial thalamic N-acetylaspartate/cytosolic choline + creatine/phosphocreatine and N-acetylaspartate/cytosolic choline levels in 11 pediatric patients with obsessive-compulsive disorder, 8 to 15 years, versus 11 case-matched healthy controls. These changes may reflect a change in N-acetylaspartate, cytosolic choline, or creatine concentrations. Therefore, using a validated phantom replacement methodology, we obtained absolute measures (mmol/L) of N-acetylaspartate, a putative marker of neuronal viability, cytosolic choline, and creatine in these subjects. A significant increase in cytosolic choline was observed in right and left medial but not lateral thalami in patients with obsessive-compulsive disorder versus controls. N-acetylaspartate and creatine did not differ significantly between case-control pairs in the medial or lateral thalamus. These findings provide new evidence of cytosolic choline abnormalities in the thalamus in pediatric obsessive-compulsive disorder.

Increased orbitofrontal cortex levels of choline in depressed adolescents as detected by in vivo proton magnetic resonance spectroscopy

Background: The frontal lobe has been implicated in the pathology of depression in adults. Through the use of magnetic resonance spectroscopy, altered brain choline levels have also been linked to the pathophysiology of affective disorders. Methods: To identify possible alterations in orbitofrontal cortex levels of cytosolic choline in adolescents with and without depression, 22 depressed and 43 control adolescents were recruited. Of those recruited, usable proton magnetic resonance spectra were acquired from a voxel in the left anterior medial frontal lobe of 17 depressed (mean age 15.8 ± 1.6) and 28 healthy adolescents (mean age 14.5 ± 1.7). Results: Orbitofrontal cytosolic choline/creatine (Cho/Cr) ratios ( p = .032) and cytosolic choline/ N-acetyl aspartate (Cho/NAA) ratios ( p = .043) were significantly higher in the depressed subjects than in the control subjects. There were no significant differences between depressed and control subjects in gray or white matter content within the voxel. Conclusions: These findings suggest that brain cytosolic choline may be increased in depressed adolescents in comparison with control subjects and independent of a corresponding structural change. These results are consistent with similar, previously reported findings in adults and suggest that depression in adolescents is associated with alterations in orbitofrontal metabolism.

Basal ganglia choline levels in depression and response to fluoxetine treatment: An in vivo proton magnetic resonance spectroscopy study

We have investigated proton magnetic resonance spectra of the basal ganglia in 41 medication-free outpatients with major depression, prior to starting an 8-week standardized trial of open-label fluoxetine, and 22 matched comparison subjects. Upon completing the trial, depressed subjects were classified as treatment responders (n = 18) or nonresponders (n = 23), based on changes in the Hamilton Depression Rating Scale. Depressed subjects had a lower area ratio of the choline resonance to the creatine resonance (Cho/Cr) than comparison subjects. This statistically significant difference between the depressed subjects and comparison subjects was more pronounced in the treatment responders than in the nonresponders. There were no differences in the relative volumes of gray matter or white matter in the voxel used for proton spectroscopy in depressed subjects relative to comparison subjects. These results are consistent with an alteration in the metabolism of cytosolic choline compounds in the basal ganglia of depressed subjects and, in particular, those who are responsive to fluoxetine.

Differential effect of CDP-choline on brain cytosolic choline levels in younger and older subjects as measured by proton magnetic resonance spectroscopy.

Phosphatidylcholine (PtdCho), which is essential for membrane integrity and repair, is reduced in brain cell membranes with age. Evidence from both animal and in vitro studies indicates that cytidine 5' diphosphate choline (CDP-choline) can increase the synthesis of PtdCho; however, the effect of CDP-choline on brain choline metabolism has not previously been studied in human subjects. In this study, in vivo proton magnetic resonance spectroscopy (1H-MRS) was used to measure brain levels of cytosolic, choline-containing compounds before and after single oral doses of CDP-choline. Three hours after dosing, plasma choline increased similarly in younger (mean age 25 years) and older subjects (mean age 59 years). However, while the choline resonance in brain increased by 18% on average in younger subjects, it decreased by almost 6% in older subjects (P = 0.028). These results may be explained by a previously observed decrease in brain choline uptake, but not cytidine uptake, in older subjects. Additional intracellular cytidine following the administration of CDP-choline should lead to the increased incorporation of choline already present in brain into membrane PtdCho, which is not MRS-visible, consequently lowering the brain choline resonance below that of pre-treatment values. These results suggest that the cytidine moiety of CDP-choline stimulates phosphatidylcholine synthesis in human brain cell membranes in older subjects.

Decreased Brain Choline Uptake in Older Adults

To test the hypothesis that uptake of circulating choline into the brain decreases with age, because alterations in metabolism of choline may be a factor contributing to age-related degenerative changes in the brain. Cohort comparison in younger and older adults. Subjects were chosen consecutively from lists of healthy volunteers screened by medical and psychiatric interviews and laboratory tests. Younger adults (n = 12) were between the ages of 20 and 40 years (mean age, 32 years), and older adults (n = 16) were between the ages of 60 and 85 years (mean age, 73 years). After fasting overnight, subjects received choline, as the bitartrate, to yield free choline equal to 50 mg/kg of body weight. Blood was drawn for determination of plasma choline concentration by high-performance liquid chromatography, and proton magnetic resonance spectroscopy (1H-MRS) was performed to determine the relative concentration of cytosolic choline-containing compounds in the brain at baseline and after ingestion of choline. Plasma choline and cytosolic choline-containing compounds in the brain, estimated as the ratio of the choline resonance to the creatine resonance on 1H-MRS scans of the basal ganglia, were compared following blinded analyses of data from subject cohorts studied at baseline and 3 hours after choline ingestion. Levels of plasma choline and cytosolic choline-containing compounds in brain were similar at baseline in younger and older subjects. Following ingestion of choline, plasma choline concentration increased by similar proportions (76% and 80%) in both younger and older subjects. Brain cytosolic choline--containing compounds increased substantially in younger subjects (mean increase, 60%; P < .001 vs baseline). Older subjects showed a much smaller increase in brain choline-containing compounds (mean, 16%; P < .001 vs the increase in younger subjects). Uptake of circulating choline into the brain decreases with age. Given the key role of choline in neuronal structure and function, this change may be a contributing factor in onset in late life of neurodegenerative, particularly dementing, illnesses in which cholinergic neurons show particular susceptibility to loss.

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholinergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipitate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 +/- 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with 32Pi incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 +/- 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 +/- 11% of control. Depolarization of synaptosomes with 50 microM veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 +/- 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.

Inhibition of cytosolic human forebrain choline acetyltransferase activity by phospho-l-serine: A phosphomonoester that accumulates during early stages of alzheimer's disease

There is no satisfactory explanation for the cholinergic deficit characteristic of Alzheimer's disease. We have performed a series of experiments which demonstrate that (a) an inhibitor of cytosolic human brain choline acetyltransferase is present in the cytosol of Alzheimer brain tissue, (b) human brain cytosolic choline acetyltransferase activity is inhibited by phospho-L-serine in a competitive manner. Cytosol was prepared from human forebrain or amygdala and the K m for choline and acetyl CoA of the choline acetyltransferase were 750 μM and 12.5 μM, respectively. Phospho-L-serine was found to be a competitive inhibitor of this enzyme with respect to choline but not with respect to acetyl CoA with a Ki 750 μM for the human forebrain and 3 μM for human amygdala. These concentrations of phospho-L-serine are present in brain tissue at early stages of Alzheimer's disease. Several other phosphomonoesters and phosphodiesters that are increased in Alzheimer's disease were either less inhibitory or without effect. The addition of heat denatured and non-heat denatured cytosol from Alzheimers forebrain inhibited the choline acetyltransferase activity present in control human brain cytosol. The inhibitory activity of the Alzheimers cytosol was retained in TCA deproteinized samples and removed by dialysis or by alkaline phosphatase treatment. Dialysis of the cytosol increased the choline acetyltransferase activity of 5 of 8 Alzheimer's disease samples from 21 to 118% with p values of < 0.025 or < 0.001, respectively. These observations provide evidence that an endogenous non-proteinaceous, dialyzable, phosphomonoester, present in Alzheimers brain inhibits the choline acetyltransferase of both control and Alzheimers brain. Therefore, an alteration in membrane phospholipid metabolism could secondarily contribute to the hypocholinergic state observed in Alzheimer's disease.

Basal synthesis of acetylcholine in hippocampal synaptosomes is not dependent upon membrane-bound choline acetyltransferase activity

Choline acetyltransferase, the enzyme which catalyses the formation of acetylcholine within cholinergic nerve terminals, exists in both cytosolic and membrane-associated subcellular pools. In the present study, alteration in nerve terminal Cl − homeostasis was used as an experimental tool to elucidate the role of membrane-bound choline acetyltransferase in regulation of the biosynthesis of acetylcholine in rat hippocampal synaptosomes under basal or resting conditions. Reduction of extracelluar Cl − concentration from 131 to 48 mM through iso-osmotic replacement with isethionate ions produced a selective decrease, to approximately 50% of control, of nerve terminal membrane-associated choline acetyltransferase activity. Under these experimental conditions, there were no changes in the activity of cytosolic enzyme or high-affinity choline uptake, or in acetylcholine synthesis. Replacement of medium Cl − with Br - supported maintenance of synaptosomal membrane-bound choline acetyltransferase activity better than did I - or isethionate ions; high-affinity choline uptake activity and acetylcholine synthesis were affected similarly. Incubation of synaptosomes with low concentrations of the Cl - channel blockers 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (50 μM) and niflumic acid (100 μM) selectively decreased activity of the membrane-bound enzyme, with no effect on cytosolic choline acetyltransferase or high-affinity choline uptake activities. Acetylcholine synthesis was unchanged, even though membrane-bound choline acetyltransferase activity was decreased in some samples (250 μM 4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulphonic acid) to about 10% of control. Experimental manipulations designed to alter neuronal Cl − homeostasis resulted in selective changes in membrane-bound choline acetyltransferase activity, thereby allowing the first direct examination of its physiological role in regulation of acetylcholine synthesis. The activity of synaptosomal membrane-bound choline acetyltransferase was selectively decreased to about 10% of control without altering cytosolic choline acetyltransferase or high-affinity choline uptake activity. As acetylcholine synthesis did not differ from control in these experiments, it appears that membrane-bound choline acetyltransferase does not play a significant regulatory role in biosynthesis of the neurotransmitter under basal conditions.

Transport and Phosphorylation of Choline in Higher Plant Cells: Phosphorus-31 Nuclear Magnetic Resonance Studies

When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by 31P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.