Cholinergic Precursor Research Articles

Antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) affect the survival and differentiative potential of cholinergic precursors from the embryonic septal nuclei and basal forebrain: involvement of ras signaling

We investigated the effects of antioxidants N-acetylcysteine (NAC) and 2-mercaptoethanol (2-ME) on the expression of choline acetyltransferase (ChAT) in cultured cholinergic precursors from the embryonic rat septal nuclei and basal forebrain. Carboxy-dichlorofluorescein fluorescence confirmed that 2-ME inhibited intracellular oxidation. Low micromolar concentrations of 2-ME produce as much as a 12-fold increase in ChAT; this is enhanced further by inclusion of nerve growth factor (NGF). NAC effects are biphasic: 0.15 mM produces profound increases in ChAT while 1.5 mM has no effect. Immature (E16) cultures respond with increases in ChAT while more highly differentiated cultures (E18) do not. Labeling of single precursors with a lacZ-expressing retrovirus reveals that the increase in ChAT is due primarily to an increased number and size of clones, not an increase in cholinergic neurons per clone, suggesting an effect on precursor survival. Inhibition of ras farnesylation inhibits both 2-ME and NAC induction of ChAT suggesting a ras-mediated pathway. Inclusion of the MEK inhibitor PD98059 does not affect low doses of NAC, but at doses of NAC that fail to increase ChAT activity, inhibition of the pathway actually raises ChAT. Immunocytochemical investigation of the cultures indicates that cells exposed to low doses of NAC develop healthy neuronal arbors in the apparent absence of glial support. At higher concentrations of NAC, neurons were found in association with astrocytes, making contact via elaborate varicose fibers. Treatment of the cultures with PD98059 to inhibit MEK returned cultures to a ‘low-dose’ phenotype. These data suggest that redox status of basal forebrain precursors affect both their survival and differentiative potential.

Treatment of cognitive dysfunction associated with Alzheimer's disease with cholinergic precursors. Ineffective treatments or inappropriate approaches?

The observations of the loss of cholinergic function in neocortex and hippocampus in Alzheimer's disease (AD) developed the hypothesis that replacement of cholinergic function may be of therapeutic benefit to AD patients. The different approaches proposed or tested included intervention with acetylcholine (ACh) precursors, stimulation of ACh release, use of muscarinic or nicotinic receptor agonists and acetylcholinesterase (AChE) or cholinesterase (ChE) inhibition. Inhibition of endogenous ACh degradation through ChE inhibitors and precursor loading were treatments more largely investigated in clinical trials. Of the numerous compounds in development for the treatment of AD, AChE and ChE inhibitors are the most clinically advanced, although clinical trials conducted to date did not always confirm a significant benefit of these drugs on all symptom domains of AD. The first attempts in the treatment of AD with cholinergic precursors did not confirm a clinical utility of this class of compounds in well controlled clinical trials. However, cholinergic precursors most largely used such as choline and phosphatidylcholine (lecithin) were probably not suitable for enhancing brain levels of ACh. Other phospholipids involved in choline biosynthetic pathways such as CDP-choline, choline alphoscerate and phosphatidylserine clearly enhanced ACh availability or release and provided a modest improvement of cognitive dysfunction in AD, these effects being more pronounced with choline alphoscerate. Although some positive results cannot be generalized due to the small numbers of patients studied, they probably would justify reconsideration of the most promising molecules in larger carefully controlled trials.

Choline alphoscerate in cognitive decline and in acute cerebrovascular disease: an analysis of published clinical data

This paper has reviewed the documentation on the clinical efficacy of choline alphoscerate, a cholinergic precursor, considered as a centrally acting parasympathomimetic drug in dementia disorders and in acute cerebrovascular disease. Thirteen published clinical trials, examining in total 4054 patients, have evaluated the use of choline alphoscerate in various forms of dementia disorders of degenerative, vascular or combined origin, such as senile dementia of the Alzheimer's type (SDAT) or vascular dementia (VaD) and in acute cerebrovascular diseases, such as transitory ischemic attack (TIA) and stroke. Analysis has assessed the design of each study, in particular with respect to experimental design, number of cases, duration of treatment and tests used to evaluate drug clinical efficacy. Most of the ten studies performed in dementia disorders were controlled trials versus a reference drug or placebo. Overall, 1570 patients were assessed in these studies, 854 of which in controlled trials. As detected by validated and appropriate tests, such as Mini Mental State Evaluation (MMSE) in SDAT and Sandoz Clinical Assessment Geriatric (SCAG) in VaD, administration of choline alphoscerate significantly improved patient clinical condition. Clinical results obtained with choline alphoscerate were superior or equivalent to those observed in control groups under active treatment and superior to the results observed in placebo groups. Analysis stresses the clear internal consistency of clinical data gathered by different experimental situations on the drug effect, especially with regard to the cognitive symptoms (memory, attention) characterising the clinical picture of adult-onset dementia disorders. The therapeutic usefulness of choline alphoscerate in relieving cognitive symptoms of chronic cerebral deterioration differentiates this drug from cholinergic precursors used in the past, such as choline and lecithin. Three uncontrolled trials were performed with choline alphoscerate in acute cerebrovascular stroke and TIA, totalling 2484 patients. The results of these trials suggest that this drug might favour functional recovery of patients with cerebral stroke and should be confirmed in future investigations aimed at establish the efficacy of the drug in achieving functional recovery of patients with acute cerebrovascular disease.

Developmental restriction of the LIM homeodomain transcription factor Islet-1 expression to cholinergic neurons in the rat striatum.

LIM homeodomain transcription factors play crucial roles in determining diverse aspects of neuronal development both in vertebrates and invertebrates. In the present study, we studied the expression pattern of Islet-1 (Isl-1), a member of the LIM homeodomain protein family, in the rat striatum during development. The developmental expression of Isl-1 in the striatum is highly dynamic and complex in terms of spatial and temporal regulation. The reverse transcription-polymerase chain reaction and ribonuclease protection assays demonstrated that Isl-1 messenger RNA was expressed in the developing striatum. The immunocytochemical study of Isl-1 protein expression showed that there were prominent mediolateral and caudorostral Isl-1 gradients in the developing striatum. Numerous Isl-1-positive cells appeared in the medial mantle zone of the developing striatal proper, and they co-expressed the postmitotic neuronal marker, microtubule-associated protein 2. The numbers of Isl-1-positive cells were decreased from the medial to the lateral regions, so that there were only a few Isl-1-positive cells scattered in the lateral striatum. These scattered Isl-1-positive cells were doubly labeled with tyrosine kinase receptor A and choline acetyltransferase, which indicated that they were cholinergic neurons. The Isl-1 gradients were most prominent in the embryonic day 18 and 20 striatum. With increases of time, the Isl-1 gradients were gradually reduced, and the gradients disappeared by postnatal day 7. Despite the general down-regulation of striatal Isl-1, a few Isl-1-positive cells were sustained into the adult striatum in which Isl-1 was nearly exclusively expressed by all cholinergic neurons and vice versa. Our study suggests that Isl-1 is likely to be initially expressed by postmitotic cholinergic precursors and some, if not all, non-cholinergic precursors in the developing striatum. During the progression of striatal differentiation, Isl-1 is down-regulated in non-cholinergic cells, but is sustained in cholinergic cells. The developmental restriction of Isl-1 to cholinergic neurons in the striatum may represent a novel mechanism by which LIM homeodomain proteins specify specific cell types in the striatum during development.

Metrifonate: a new agent for the treatment of Alzheimer's disease.

The pharmacology, pharmacokinetics, clinical efficacy, and adverse effects of metrifonate, a long-acting cholinesterase inhibitor, are discussed. Attempts to correct the central cholinergic deficits associated with Alzheimer's disease have included administration of cholinergic precursors, cholinergic agonists, and cholinesterase inhibitors. To date, two reversible cholinesterase inhibitors-tacrine and donepezil-have been marketed. Metrifonate, an organophosphate, is converted nonenzymatically to 2,2-dichlorovinyl dimethyl phosphate (DDVP), the active enzyme inhibitor. DDVP produces irreversible inhibition of brain cholinesterase that lasts for several days; enzyme recovery is dependent on synthesis of new enzyme. Several preclinical studies have demonstrated cognition-enhancing effects of metrifonate in animals. Trials in humans have shown improvement on the Alzheimer's Disease Assessment Scale, in Mini-Mental State Examination scores, and in the Clinician's Interview-Based Impression of Change with caregiver input. Clinical improvement noted with metrifonate appears similar to that seen with other cholinesterase inhibitors. Adverse effects noted in clinical trials have been associated primarily with the gastrointestinal tract and have been mild. Metrifonate appears to be a promising agent for the treatment of the symptoms of Alzheimer's disease.

Chapter 28 Clinical approaches to dementia

Dementia—like mental retardation—is a symptom and not a disease. For the age group 50–90 years, Alzheimer's disease and vascular dementia predominate; whereas in younger patients, AIDS would lead the list in many countries. The effects of aging with a presumed disease process—such as Alzheimer's disease—make studies of dementia difficult. The cholinergic hypothesis has resulted in a variety of therapeutic attempts to increase acetylcholine in brain. Based on the precursor strategy, successfully employed in the use of L-dopa in Parkinson's disease, cholinergic precursors—such as lecithin—have been tried, but without significant success. The Food and Drug Administration (FDA) of the United States has approved tacrine (tetrahydroaminoacridine) for treatment of dementia. This approval is based primarily on a prospective double-blind, placebo-controlled study on 480 patients. The results suggest that Tacrine reverses signs of dementia in some patients with Alzheimer's disease. Further, neurochemical studies have indicated that Alzheimer's disease is not a deficiency of a single transmitter. It is found that gangliosides also have potential as neuroprotective or neurorehabilitative agents.

The pharmacological management of cognitive impairment in the demented patient

To date therapeutic compounds for cognitive impairment in dementia has been orientated to neurotransmitter manipulation by cholinergic precursors, agonists or protectors or adrenergic seratonergic or GABAnergic drugs or by agents which enhance synaptic connections and/or limit cell death.

Cholinergic mechanisms in startle and prepulse inhibition: effects of the false cholinergic precursor N-aminodeanol.

We examined the effects of cholinergic deficiency on prepulse inhibition (PPI) of the acoustic startle. Rats treated with a choline-free diet that contained the false cholinergic precursor N-aminodeanol showed great deficit in PPI. This deficit does not appear to be secondary to an increase of stereotyped behaviors. Startle threshold was also greatly reduced, as these rats startled to the 70-dB prepulse and the baseline startle amplitude was increased by 60% over the control rats. Arecoline (4 mg/kg) partially reversed the deficit in PPI. This improvement persisted beyond the period of drug treatment. On the other hand, scopolamine (1 mg/kg) reduced PPI in the control rats. These results suggest that cholinergic systems play a major role in both the elicitation and prepulse inhibition of startle.

Cholinergic mechanisms in startle and prepulse inhibition: Effects of the false cholinergic precursor N-aminodeanol.

Cholinergic mechanisms in startle and prepulse inhibition: Effects of the false cholinergic precursor N-aminodeanol.

Morphogenesis of adrenergic cells in a frog parasympathetic ganglion

The development of the parasympathetic cardiac ganglion of the frog Xenopus laevis was marked by the differentiation of a population of adrenergic small intensely fluorescent (SIF) cells. The neural crest contributes precursors for both SIF cells and the well-studied cholinergic cardiac ganglion neurons; this situation provided an opportunity to determine whether morphogenesis of the two cell types was correlated. Accordingly, we examined the initial differentiation, developmental regulation, and territorial domain of cardiac SIF cells for comparison with their cholinergic neuron neighbors. Adrenergic SIF cells were present during the time when the first cholinergic precursors were becoming postmitotic. Although SIF cells were present first, cholinergic neurons differentiated almost 16 times faster during the first week of embryonic and larval development and outnumbered SIF cells at all subsequent stages. Nonetheless, the accumulation of both cell types were correlated, since the ratio of cholinergic neurons to SIF cells remained at approximately 10 to 1 up to adult life. Early in development, SIF cells and cholinergic neurons were clustered in the sinus venosus portion of the atrium. The asymmetric distribution of cholinergic neurons within the atrium was lost but that of the SIF cells was maintained throughout life. These results identify relationships between the morphogenesis of two cell types in an autonomic ganglion and place constraints upon the cellular mechanisms that could produce the cells from their neural crest precursors.

Effects of chronic in vivo replacement of choline with a false cholinergic precursor.

Chronic administration to rats of a diet in which all choline is replaced by NADe, an unnatural choline analog, results in a classical hypocholinergic syndrome characterized by progressive loss of learning and memory, hyperkinesis, hyperreactivity and hyperalgesia. Discontinuation of the artificial diet results in rapid elimination of NADe from both free and phospholipid-bound pools in all tissues studied, but the behavioral effects recede more slowly and incompletely. These results are consistent with a model in which choline and NADe compete in both acetylcholine and phospholipid synthesis, resulting in selective vulnerability of cholinergic neurons. Histological studies are in progress to determine whether microanatomical changes are also consistent with this model.

Piracetam elevates muscarinic cholinergic receptor density in the frontal cortex of aged but not of young mice.

Chronic treatment (2 weeks) with piracetam (500 mg/kg, once daily PO) elevated m-cholinoceptor density in the frontal cortex of aged (18 months) female mice by about 30-40%, but had no effect on m-cholinoceptor density in the frontal cortex of young (4 weeks) mice. The effect of piracetam on m-cholinoceptor density as determined by the specific binding of tritiated QNB was not affected by concomitant daily treatment with either choline (200 mg/kg) or scopolamine (4 mg/kg). It is concluded that the effect of piracetam on m-cholinoceptor density could explain the positive effects which have been reported for combinations of cholinergic precursor treatment with piracetam on memory and other cognitive functions in aged experimental animals and patients and could also represent part of the possible mechanism of action of piracetam alone.

Cholinergic hypothesis of human memory: Review of basic and clinical studies

AbstractRecent neurochemical data from autopsy and biopsy studies of Alzheimer patients lends strong support for a selective impairment of cholinergic functioning. A number of pharmacological trials are described testing the effects of cholinergic agonists and antagonists on cognitive functions of both young normal volunteers as well as geriatric, demented patients. Paradigms included prelearning and postlearning pharmacological manipulations as well as testing the effect of a combination of a cholinergic precursor (lecithin) plus an anticholinesterase (tetrahydroaminoacridine). The results indicated great variability of responses among subjects, with mild‐to‐moderate enhancement of memory recall in specific subgroups of normal controls and demented patients. Further research should be aimed at developing reliable in vivo cholinergic markers and validation of these measures as response predictors of specific cholinergic treatments in patients with dementia.

Attenuation of pilocarpine-induced hypothermia in response to chronic administration of choline.

Chronic treatment of rats with choline caused a decrease in the hypothermic response to pilocarpine. The action of choline on the muscarinic receptors is consistent with electrophysiological and binding studies, supporting a direct muscarinic action for choline. Administration of direct muscarinic agonists has been shown to cause a decrease in the number of muscarinic receptors. Thus, the long-term use of cholinergic precursors could have some adverse effects on central cholinergic functioning.

The use of cholinergic precursors in neuropsychiatric diseases

Preclinical data suggest that cholinergic precursors such as choline or lecithin, increase levels of acetylcholine in specific brain structures, and under certain conditions may enhance cholinergic neurotransmission. A variety of neuropsychiatric diseases including tardive dyskinesia. Huntington's chorea, ataxias, Tourette's syndrome, schizophrenia, affective illness, and senile dementia of the Alzheimer type, has been implicated with a general underactivity of central cholinergic mechanisms. Recent studies have investigated the possibility that cholinergic precursor loading strategies may provide viable treatments for these disorders of presumed cholinergic underactivity. Extensive data demonstrate that the symptoms of tardive dyskinesia can be reduced by choline or lecithin, whereas investigations in other disorders have met with mild success, at best, or are still in preliminary stages. Further controlled studies with choline or lecithin using broader dose ranges, longer durations of treatment, and concomitant administration of agents which may increase the release of acetylcholine are warranted.

Piracetam in the treatment of cognitive impairment in the elderly

AbstractReisberg, B., S.H. Ferris, M.K. Schneck, J. Corwin, P. Mir, E. Friedman, K.A. Sherman, M. McCarthy, and R.T. Bartus: Piracetam in the treatment of cognitive impairment in the elderly. Drug Dev. Res. 2: 475–480, 1982.Piracetam (Nootropil, 2‐oxopyrrolidone acetamide) has been extensively investigated for the treatment of cognitive impairment. Initial studies on normal subjects and patients with mild or moderate cognitive decline have been somewhat encoruaging. Accordingly, we conducted a further evaluation of the effects of piracetam in the treatment of elderly outpatients 60 to 85 years of age with mild to moderate memory impairment consistent with a diagnosis of Primary Degenerative Dementia (PDD). In our first study, we examined the effects of piracetam in 20 patients. All patients received 7.2 g of piracetam and placebo for 4 weeks in accordance with a double‐blind, randomized treatment order, crossover design with 1‐week washout periods prior to each crossover period. Hence, the total study period for each patient was 10 weeks (1‐4‐1‐4). An analysis of 43 psychometric measures revealed significant improvement (P < 0.05) in only three measures, all favoring the treatment condition. Recent findings support a rationale for examining the effects of piracetam in conjunction with cholinergic precursors in patients with cognitive decline. In our second study we conducted a 1‐week open trial of 1.6 g of piracetam t.i.d. in conjunction with 3 g of choline cholride t.i.d. in 15 patients. Four patients were rated as clinically improved. These “responders” were all subjects with moderate cognitive impairment. The responders showed much higher RBC choline levels than the nonresponders, both at baseline and during treatment. We conclude that the present evidence indicates that the effects of piracetam treatment alone in elderly outpatients with mild to moderate congnitive decline are subtle and not of proven clinical significance. However, studies of longer duration and of piracetam in combination with other agents may eventually show genuine clinical utility.

Memory Deficits in Aged Cebus Monkeys and Facilitation With Central Cholinomimetics

Cebus monkeys of 3 different age groups were trained to perform an automated behavioral task (delayed response), intended to measure recent memory ability. In in initial study, the aged monkeys (18 years and older) exhibit prprogressively greater performance impairments (relative to young monkeys) as they were required to remember the location of a visual stimulus for increasingly longer durations (0 to 20 sec). This deficits replicated previously published results from aged Rhesus monkeys and appeared similar to the primary memory deficits reported in elderly humans and demented patients. In subsequent studies, the effects of three different cholinomimetics were evaluated for their ability to improve the aged monkey's performance on this task. Each monkey was tested under several acute doses of the cholinergic precursor, choline, the anticholinesterase, physostigmine, and the cholinergic muscarinic receptor agonist, arecoline. The results revealed clear differences in the ability of these drugs to improve performance on this task. Choline exerted no apparent effects in the aged monkeys at any dose tested. Physostigmine clearly enhanced performance in certain aged monkeys, but the optimal dose varied dramatically between subjects, replicating previously published results with aged Rhesus monkeys and humans. Arecoline produced clear improvement within a restricted dose range, with little variation in optimal dose between subjects. In addition to demonstrating differences in the effects of different cholinomimetics on memory performance in aged primates, these data also suggest a possible rationale for future investigations. Assuming that each of these drugs primarily affected cholinergic function in the manner conventionally attributed, these data suggest that, within the cholinergic system, the more directly one stimulates the receptor, the more one might expect robust and consistent effects on memory performance in aged subjects.