Cholinergic Neurons In Alzheimer's Disease Research Articles

An arginine-rich peptide inhibits AChE: template-based design, molecular modeling, synthesis, and biological evaluation.

Life expectancy is growing especially in developed countries. In this regard, aging-associated diseases such as Alzheimer's disease (AD) are more common. Multi interconnected pathological factors involved in AD demand multi-target therapeutics. AChE, as a well-known target in AD, decreases the acetylcholine (ACh) in cholinergic synapse and, besides, increases the rate of amyloid-beta (Aβ) aggregation. To block the destructive effects of AChE on cholinergic neurons in AD, we designed a peptidic inhibitor of the peripheral anionic site (PAS). The PAS plays a crucial role to attract and direct the ACh to the enzyme active site and increase the rate of Aβ aggregation by changing the folding state. We utilized the template-based approach in combination with molecular docking, molecular dynamic simulation, and data mining to design a peptide library. Scoring was performed according to binding energy and the interaction profile of AChE inhibitors. The best candidate (p8, RMLRTTRY) was synthesized using solid-phase peptide synthesis, purified by RP-HPLC, and identified by ESI-MS. The inhibitory effect of p8 on AChE was 102.2 ± 15.2μM. The kinetic and molecular modeling studies indicated the mixed inhibition mechanism for p8. The Arg residues in p8 had an essential role in binding to PAS.

The NGF Metabolic Pathway: New Opportunities for Biomarker Research and Drug Target Discovery : NGF Pathway Biomarkers and Drug Targets.

Recent research has demonstrated that degeneration of the basal forebrain cholinergic system, far from being a mere downstream mediator of Alzheimer's disease (AD) symptoms, may play a disease-aggravating role in the continuum of AD pathology. The search for novel biomarkers of the cholinergic deficit in AD and novel therapeutic targets for the sustenance of the basal forebrain cholinergic system has therefore taken on more urgency. A novel model that explains the preferential vulnerability of basal forebrain cholinergic neurons in AD as the result of pathological alterations to nerve growth factor (NGF) metabolism offers an integrated investigative platform for the development of such biomarkers and therapeutics. By positing a reciprocal trophic interaction between the basal forebrain and its target tissues, this model can also explain the disease-modifying nature of the cholinergic deficit in AD and can incorporate other key factors in basal forebrain cholinergic degeneration, including NGF receptor changes and retrograde transport deficits in AD. This chapter will focus on the potential of NGF metabolic pathway biomarkers in AD as well as therapeutic targets to correct NGF metabolic deficits, aiding the development of novel pro-cholinergic therapeutics.

Effect of the Biphenyl Neolignan Honokiol on Aβ42-Induced Toxicity in Caenorhabditis elegans, Aβ42 Fibrillation, Cholinesterase Activity, DPPH Radicals, and Iron(II) Chelation

The biphenyl neolignan honokiol is a neuroprotectant which has been proposed as a treatment for central nervous system disorders such as Alzheimer's disease (AD). The death of cholinergic neurons in AD is attributed to multiple factors, including accumulation and fibrillation of amyloid beta peptide (Aβ) within the brain; metal ion toxicity; and oxidative stress. In this study, we used a transgenic Caenorhabditis elegans model expressing full length Aβ42 as a convenient in vivo system for examining the effect of honokiol against Aβ-induced toxicity. Furthermore, honokiol was evaluated for its ability to inhibit Aβ42 oligomerization and fibrillation; inhibit acetylcholinesterase and butyrylcholinesterase; scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals; and chelate iron(II). Honokiol displayed activity similar to that of resveratrol and (-)-epigallocatechin gallate (EGCG) in delaying Aβ42-induced paralysis in C. elegans, and it exhibited moderate-to-weak ability to inhibit Aβ42 on-pathway aggregation, inhibit cholinesterases, scavenge DPPH radicals, and chelate iron(II). Moreover, honokiol was found to be chemically stable relative to EGCG, which was highly unstable. Together with its good drug-likeness and brain availability, these results suggest that honokiol may be amenable to drug development and that the synthesis of honokiol analogues to optimize these properties should be considered.

Treatment of beta amyloid 1-42 (Aβ(1-42))-induced basal forebrain cholinergic damage by a non-classical estrogen signaling activator in vivo.

In Alzheimer’s disease (AD), there is a loss in cholinergic innervation targets of basal forebrain which has been implicated in substantial cognitive decline. Amyloid beta peptide (Aβ1–42) accumulates in AD that is highly toxic for basal forebrain cholinergic (BFC) neurons. Although the gonadal steroid estradiol is neuroprotective, the administration is associated with risk of off-target effects. Previous findings suggested that non-classical estradiol action on intracellular signaling pathways has ameliorative potential without estrogenic side effects. After Aβ1–42 injection into mouse basal forebrain, a single dose of 4-estren-3α, 17β-diol (estren), the non-classical estradiol pathway activator, restored loss of cholinergic cortical projections and also attenuated the Aβ1–42-induced learning deficits. Estren rapidly and directly phosphorylates c-AMP-response–element-binding-protein and extracellular-signal-regulated-kinase-1/2 in BFC neurons and restores the cholinergic fibers via estrogen receptor-α. These findings indicated that selective activation of non-classical intracellular estrogen signaling has a potential to treat the damage of cholinergic neurons in AD.

Neurobiology of Arousal and Sleep: Updates and Insights Into Neurological Disorders

Brain activation during wakefulness is sustained by multiple arousal systems. Dysfunction of one or more arousal systems is a feature of neurological disorders associated with hypersomnolence and/or sleep-wake cycle disturbance. Narcolepsy, Alzheimer’s disease (AD), Parkinson’s disease (PD), and traumatic brain injury appear to involve hypocretin (HCT) and possibly histamine insufficiency as a mechanism related to excessive daytime sleepiness. Loss of cholinergic neurons in AD and of dopamine neurons in PD contributes to sleep-wake disturbance. GABAergic neurons in the preoptic hypothalamus and rostral medulla promote sleep through inhibition of arousal systems. Pathology of preoptic sleep regulatory circuits is correlated with sleep disturbance in AD. An unidentified endogenous somnogen that potentiates the actions of gamma-aminobutyric acid (GABA) is present in the cerebrospinal fluid (CSF) of patients with primary hypersomnia. Descending pathways from the dorsal lateral pons to the ventral medulla and spinal cord are responsible for the inhibition of spinal motoneurons during rapid eye movement (REM) sleep and are implicated in human REM sleep behavior disorder.

Human Neural Stem Cells Genetically Modified to Express Human Nerve Growth Factor (NGF) Gene Restore Cognition in the Mouse with Ibotenic Acid-Induced Cognitive Dysfunction

Alzheimer's disease (AD) is characterized by degeneration and loss of neurons and synapses throughout the brain, causing the progressive decline in cognitive function leading to dementia. No effective treatment is currently available. Nerve growth factor (NGF) therapy has been proposed as a potential treatment of preventing degeneration of basal forebrain cholinergic neurons in AD. In a previous study, AD patient's own fibroblasts genetically modified to produce NGF were transplanted directly into the brain and protected cholinergic neurons from degeneration and improved cognitive function in AD patients. In the present study, human neural stem cells (NSCs) are used in place of fibroblasts to deliver NGF in ibotenic acid-induced learning-deficit rats. Intrahippocampal injection of ibotenic acid caused severe neuronal loss, resulting in learning and memory deficit. NGF protein released by F3.NGF human NSCs in culture medium is 10-fold over the control F3 naive NSCs at 1.2 µg/10(6) cells/day. Overexpression of NGF in F3.NGF cells induced improved survival of NSCs from cytotoxic agents H2O2, Aβ, or ibotenic acid in vitro. Intrahippocampal transplantation of F3.NGF cells was found to express NGF and fully improved the learning and memory function of ibotenic acid-challenged animals. Transplanted F3.NGF cells were found all over the brain and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs overexpressing NGF improve cognitive function of learning-deficit model mice.

P-455 - Analysis of the effect of galantamine in the treatment of mild to moderate Alzheimer's disease (AD)

Alzheimer's disease is a progressive neurodegenerative disorder in which the patient experiences progressive loss of cognitive and functional abilities and concomitant behavioral disturbances. While all acetylcholinesterase inhibitors enhance brain activity by inhibiting the breakdown of acetylcholine, Galantamine differs from the other cholinesterase inhibitors in that it allosterically modulates nicotinic acetylcholine receptors. Allosteric modulation of presynaptic nicotinic receptors is thought to cause an increase in the release of several neurotransmitters. These actions help to compensate for the decreased release of acetylcholine associated with the loss of cholinergic neurons in AD. Subjects were diagnosed with mild to moderate probable AD based on the criteria set by the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer Disease and Related Disorders Association, namely a screening score of 10–24 on the Mini-Mental State Examination (MMSE) and a score of ≥ 18 on 11-item cognitive subscale of the Alzheimer Disease Assessment Scale (ADA - cog11). One hundred subjects received 1 (one) capsule of Galantamine twice per day in the morning and evening for 6 (six) months. The administration of the Galantamine was associated with significant greater cognitive benefits and significant better maintenance of daily activities and was generally safe and has proven better tolerability.

Neuropathological Changes in Adult and Old Rats Following Early Post-Natal Iron Administration

acetylcholine esterase (AChE) activity. Evidence for cholinergic dysfunction in early stages of Alzheimer’s disease (AD) or mild cognitive impairment (MCI) is inconclusive, and human studies of the relationship of cholinergic function with neuropsychological performance in vivo are rare. Methods: We included 21 healthy controls, 20 MCI and 15 mild AD patients. A comprehensive neuropsychological battery was administered to indicate the general cognitive function and to define performance of verbal and non-verbal memory, language, executive function and visuoconstructive abilities. [11C]-MP4A PET was applied in all subjects, parametric images of tracer hydrolysis rate constant k3 were generated and analysed using standard atlas regions. After principal component analysis (PCA) of k3 values correlational analysis with neuropsychological test results was performed. Results: The mean global k3 values of MCI (0.076 6 0.011 min-1) and AD patients (0.066 6 0.009 min-1) were significantly lower than that of controls (0.084 6 0.006 min-1) (p < 0.05 and p < 0.001, respectively) with the temporal regions showing the strongest decline in both patient groups. PCA revealed three main components representing frontal and parietal regions (PC1), temporal regions (PC2) and hippocampus and amygdala (PC3). PC 2 could separate all three diagnostic groups. Significant correlations in the whole group between PC2 and neuropsychological performances were found, most strongly with impaired verbal and visual memory function as well as impaired working memory, but also with deficient visuo-spatial and executive functions. The association of verbal and non-verbal memory with the ‘‘temporal’’ PC 2 was confirmed in MCI patients. This is in line with neuropathological studies showing the greatest and most consistent loss of cholinergic neurons in AD in the posterior part of the nucleus basalis Meynert (nbM-Ch4p), which primarily sends cholinergic projections to the temporal cortex (Geula and Mesulam, 1999). Conclusions: Cholinergic dysfunction in the temporal lobe is an early hallmark in MCI and mild AD and it is associated with impaired neuropsychological function.

Coexistences of insulin signaling-related proteins and choline acetyltransferase in neurons

Type 2 diabetes recently has been identified as a risk factor for developing Alzheimer's disease (AD). The main reason for this appears to be insulin signaling failure in the brain. Furthermore, cholinergic neurons are particularly affected in the brains of AD patients. The aim of the present study is to investigate if insulin signaling-related proteins are co-located with cholinergic neuron in the CA1 region of hippocampus of mice, which could explain the early loss of cholinergic neurons in AD. Using immunohistochemistry, the insulin signaling-related proteins, such as insulin receptor (InsR), insulin receptor substrate-1 (IRS-1), protein kinase B (PKB, also named Akt), glycogen synthatase kinase-3bteta (GSK-3bteta) and insulin-degrading enzyme (IDE) were analysed. Choline acetyltransferase (ChAT) was selected as a marker of cholinergic neurons. In the CA1 region of hippocampus of mice, several of the insulin signaling-related proteins we had chosen are co-located with ChAT, and most double immunoreactive positive cells were pyramidal cells. The coexistences indicated that the insulin signaling may play an important part in the activities of cholinergic neurons, and the impairment of the pathway may be important in the mechanisms that underlie neurodegeneration in AD.

P3-402: Age-related loss of calbindin identifies basal forebrain cholinergic neurons destined to degenerate in Alzheimer's disease

The reasons for the vulnerability of select neuronal populations in Alzheimer's Disease (AD) remain unknown. We have used the basal forebrain cholinergic neurons (BFCN), which are vulnerable to degeneration early in the course of AD, to determine of the causes of selective neuronal vulnerability. We have observed a selective loss of the calcium binding protein calbindin-D28K from the human BFCN in the course of normal aging. The calcium dysregulation caused by the loss of calbindin may contribute to the loss of BFCN in AD. In the present set of experiments, we investigated whether the BFCN which degenerate in AD are those which lose calbindin in the course of aging. Immunohistochemical methods were used in postmortem brains from young (<65 years), old and AD cases. Cell counting techniques were used to determine the numbers of immunoreactive neurons. The number of choline acetyltransferase (ChAT) and low affinity neurotrophin receptor (p75NTR) immunoreactive BFCN remained constant in old individuals when compared with the young and displayed a significant decrease in AD. In contrast, the number of calbindin-positive BFCN showed a significant decrease in normal aged individuals when compared with the young and remained constant in AD. The percentage of calbindin-positive BFCN in the aged was significantly lower when compared with the young. Of great interest, the percentage of the remaining BFCN in AD which contained calbindin immunoreactivity displayed a significant increase when compared with the normal aged, indicating that the majority of surviving BFCN in AD are calbindin-positive. Western blot analysis showed no increase in the total levels of calbinidin protein in AD basal forebrain, suggesting that the maintained calbindin immunoreactivity in AD BFCN is not due to reactive upregulation. These observations indicate that the loss of cholinergic neurons in AD occurs preferentially in the BFCN which lose their calbindin in the course of normal aging and that the BFCN which contain calbindin survive the neurodegenerative process. It is likely that loss of calbindin during aging deprives the BFCN from the calcium buffering capacity of this protein, leaving them vulnerable to degeneration due to calcium toxicity in AD.

MR features of the substantia innominata and therapeutic implications in dementias

We measured the thickness of the substantia innominata using magnetic resonance imaging in 122 patients with Alzheimer's disease (AD), 31 patients with dementia with Lewy bodies (DLB) and 34 patients with vascular dementia (VaD), and examined the correlates of cognitive response to donepezil. Although all dementia groups showed significant atrophy of the substantia innominata compared to 28 age-matched controls, atrophy was greater in the DLB group, but less in the VaD group than the AD group. Mini-Mental State Examination score changes at 12 weeks after donepezil administration inversely and significantly correlated with the thickness of the substantia innominata in patients with AD ( n = 103, r = −0.43, p < 0.0001) and in patients with DLB ( n = 24, r = −0.57, p < 0.01), but not in patients with VaD ( n = 12, r = −0.22, p > 0.1). There may be some differences in cholinergic impairment among AD, DLB and VaD, reflecting cholinergic neuropathology. Clinical response to cholinergic therapy may be partly attributable to damaged cholinergic neurons in AD and DLB, but not in VaD, suggesting differences in the therapeutic implication of cholinergic system degeneration.

Apoptotic signals within the basal forebrain cholinergic neurons in Alzheimer's disease

A relatively early and substantial loss of basal forebrain cholinergic neurons is a constant feature of Alzheimer's disease (AD). However, the mechanisms that contribute to the selective vulnerability of these neurons are not fully delineated. In the present series of experiments, we determined the possible contribution of apoptotic processes and other pathologic cascades to the degeneration of the cholinergic neurons of the nucleus basalis of Meynert (NBM) in AD. In contrast to neurons in the frontal cortex which showed prominent DNA fragmentation as detected by the TUNEL method, no DNA fragmentation was observed within the NBM in any of the AD or normal brains. Similarly, immunoreactivity for the apoptotic signals Fas, Fas-ligand, Bax, Bcl-x, caspase-8, caspase-9 and caspase-3 was absent from the NBM of AD and control brains. In contrast, a substantial subpopulation of cholinergic neurons within the NBM in AD displayed prominent immunoreactivity for the apoptotic signal Fas-associated death domain (FADD) in the form of tangles. FADD immunoreactivity was also present in dystrophic neurites. FADD-positive tangle-like structures were localized in neurons which contained immunoreactivity for the cholinergic marker choline acetyltransferase (ChAT) and the low affinity neurotrophin receptor p75 NTR. While many of the NBM cholinergic neurons in control brains contained immunoreactivity for the calcium binding protein calbindin-D 28K (CB), the NBM neurons in AD displayed a substantial loss of CB immunoreactivity. Importantly, most of FADD-immunoreactive cholinergic neurons were devoid of CB immunoreactivity, and, conversely, most CB-positive cholinergic neurons had no FADD immunoreactivity. FADD immunoreactivity within the basal forebrain was colocalized with phosphorylated tau immunoreactive tangles and dystrophic neurites. In contrast, FADD immunoreactivity did not appear to be related to the primarily diffuse amyloid-β deposits intermingled between cholinergic neurons in AD NBM. Finally, many CD68-positive microglia were observed surrounding the NBM cholinergic neurons in AD. In conclusion, the findings of the present study indicate that, while the FADD apoptotic signaling pathway may be triggered within the basal forebrain cholinergic neurons in AD, the apoptotic cascade is most likely aborted as no DNA fragmentation was detected and the executioner caspase-3 was not up-regulated within these neurons. The findings also suggest possible relationships between loss of CB, FADD expression and phosphorylation of tau within the basal forebrain cholinergic neurons in AD.

Applications of Laser Capture Microdissection in the Study of Neurodegenerative Disease

Laser capture microdissection (LCM) is a new technology that is becoming increasingly important for studies of neurodegenerative disorders. A characteristic feature of all neurodegenerative diseases is “selective vulnerability.” In each of the disorders, there is selective degeneration of particular types of neurons, with relative preservation of much of the rest of the brain. Familiar examples are the selective degeneration of dopaminergic neurons in Parkinson disease, the striking depletion of basal forebrain cholinergic neurons in Alzheimer disease, the selective atrophy of the caudate nucleus in Huntington disease, and the loss of spinal motor neurons in amyotrophic lateral sclerosis. Closer examination reveals an even more finegrained pattern of neuronal injury. For example, in Parkinson disease there is striking depletion of dopaminergic neurons in the ventral tier of the substantia nigra pars compacta, with very little injurytodopaminergicneuronslocatedonlyafewmillimetersawayinthedorsaltierofthenucleus. In Huntington disease, there is an exquisite degree of selectivity among the different types of neurons found within the caudate and putamen, with striking depletion of the medium spiny projection neurons, especially those projecting to the globus pallidus, in contrast to preservation of the intermingled interneurons. These characteristic patterns of injury are evident even in genetically determined forms of neurodegenerative disease where the fundamental defect can be shown to be present in all neurons. Understanding the basis for these patterns of neuronal loss may provide important insight into the mechanisms of neurodegeneration. Selectivevulnerabilitycreatesdifficulttechnicalchallengesforinvestigatorsattempting to apply modern molecular methods to the study of disease. Many of the currentmethodsforanalyzingDNA,messengerRNA(mRNA)andproteinsrelyonhomogenization and extraction of the chemical elements of interest. Until recently,moststudiesofthisnaturehaverelied on studies of anatomical regions at least several millimeters in size, and often much larger. These areas are large enough that they unavoidably encompass both affected and unaffected neuronal populations. They also include a large component of glial structures, which may have their own distinct role in and responsetoneurodegeneration.Thedataobtained by homogenization of such heterogeneous samples are often difficult to reconcile with alterations in the biology ofspecificpopulationsofcomponentcells. Researchers can use LCM as an accessible method for separating the different cell types present in a brain region, and are able to study the biology of homogeneous cell populations with specific characteristics.

4-Hydroxynonenal, a product of lipid peroxidation, damages cholinergic neurons and impairs visuospatial memory in rats.

The mechanisms that underlie cholinergic neuronal degeneration in Alzheimer disease (AD) are unclear, but recent data suggest that oxidative stress plays a role. We report that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, damages and kills basal forebrain cholinergic neurons when administered intraparenchymally. Examination of Nissl-stained brain sections following unilateral HNE infusion revealed widespread neuronal loss in basal forebrain ipsilateral to the injection, but not on the contralateral side. Levels of choline acetyltransferase activity and immunoreactivity in the ipsilateral basal forebrain and hippocampus were significantly reduced by 60-80% seven days following HNE administration. Performance in Morris water maze tasks of visuospatial memory was severely impaired in a dose-dependent manner seven days following bilateral administration of HNE. Bilateral infusion of FeCl2 (an inducer of membrane lipid peroxidation) into the basal forebrain caused neuron loss and decreased choline acetyltransferease immunoreactivity and deficits in visuospatial memory. Additionally, FeCl2 infusion increased HNE immunoreactivity, implicating HNE in iron-induced oxidative damage. Because recent studies have demonstrated HNE adducts in degenerating neurons in AD brain, the present findings suggest a role for HNE in damage to cholinergic neurons in AD.

Cholinesterases in Alzheimer’s disease and Cholinesterase inhibitors in Alzheimer therapy

The central cholinergic neurons are known to be closely linked to intellectual functions such as memory and cognition. Alzheimer’s disease (AD) is accompanied by a substantial loss of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities associated with cholinergic and cholinoceptive neurons in several brain areas, especially in the cortex. Several reports suggest an altered form of AChE in AD. It may differ in its pH optimum and its responses to inhibitors, it may have lost its peripheral site and it has an anomalous molecular form as revealed by isoelectric focusing. All of these features point to the abnormality of the affected cholinergic neurons in AD.

Human Melanocytes as a Model System for Studies of Alzheimer Disease

The aging process leads to increased vulnerability to injury and disease, resulting in a decline in 1 or more organ systems that is incompatible with life. One of the most devastating age-associated neurodegenerative disorders, Alzheimer disease, is characterized by neuronal loss and the extracellular deposition in the brain of beta-amyloid peptide, which is presumed to be causally related. Using cultured neural crest-derived cutaneous melanocytes, we find that in the presence of beta-amyloid, melanocytes, like neurons, undergo programmed cell death (apoptosis). Nerve growth factor, which has been reported to attenuate the loss of cholinergic neurons in Alzheimer disease, protects melanocytes from apoptosis induced by beta-amyloid. Moreover, beta-amyloid is a ligand for the 75-kD transmembrane neurotrophin receptor that belongs to the family of apoptotic receptors that generates a cell-death signal on activation. Our data suggest that neuronal death in Alzheimer disease is mediated by the interaction of beta-amyloid with the 75-kD neurotrophin receptor. Human melanocytes provide a valuable in vitro model for studies of Alzheimer disease and for development of potential therapies.

Neurotrophin receptors and selective loss of cholinergic neurons in Alzheimer disease.

The most consistent neuropathological finding in Alzheimer disease (AD) is the loss of cholinergic neurons of the nucleus basalis of Meynert (NbM). Using immunohistochemistry, we have previously shown that cholinergic neurons located in the ventral striatum were affected, whereas those of the caudate nucleus, putamen, and mesencephalon were spared. Since cholinergic neurons that degenerate in AD are sensitive to NGF and those that are spared are not, it has been hypothesized that the loss of neurotrophins receptors may play a role in the death of cholinergic neurons in AD. Using immunohistochemistry, we have detected the presence of TrkA on most cholinergic neurons from the NbM, on some from those of the striatum, but not on those of the mesencephalon in the human brain. In AD patients, the number of neurons that expressed TrkA was markedly decreased in the NbM very likely as a consequence of cholinergic neuronal loss. In the striatum, despite the loss of high-affinity NGF binding previously reported, no loss of TrkA was observed. Taken together, these results suggest a decreased expression of NGF receptors on the striatal cholinergic neurons in AD. This loss may contribute, when it reaches a crucial threshold, to the death of cholinergic neurons occurring in AD.

Beta-amyloid-related peptides inhibit potassium-evoked acetylcholine release from rat hippocampal slices

The 4 kDa beta-amyloid (A beta) protein, a major component of cerebral and cerebrovascular plaques in Alzheimer's disease (AD), is derived from the proteolytic cleavage of a larger, membrane-bound precursor, the A beta precursor protein (APP). Until recently, it was assumed that an aberrant AD-specific proteolysis generated A beta peptides, which subsequently could initiate and/or contribute to the pathological cascade leading to plaque formation and losses of selected neuronal populations, including basal forebrain cholinergic neurons that provide major inputs to the hippocampus and neocortex. However, the recent detection of soluble A beta fragments in the plasma and CSF of normal individuals, as well as in the conditioned media of cultured brain cells, suggests a role for A beta-related peptides in normal brain functions. Taking into consideration the reported toxic properties of A beta and the preferential vulnerability of basal forebrain cholinergic neurons in AD, we investigated the possible effects of A beta-related peptides on the release of endogenous acetylcholine (ACh) from rat brain slices. A beta 1-28, in a concentration-dependent manner (10(-12)-10(-8) M), potently inhibited K(+)-evoked ACh release from hippocampal slices. The inhibition of ACh release was fully reversible and was observed using other A beta-related peptides such as A beta 1-42, A beta 1-40, and A beta 25-35, but not with the scrambled, reverse, or all D-isomer A beta-peptide sequences, indicating that the effect of A beta on ACh release is mediated via a stereoselective mechanism. Tetrodotoxin (10 microM) failed to alter the effect of A beta 1-28 on ACh release, which suggests the lack of involvement of voltage-dependent Na+ channels. Except for the hippocampal formation, the inhibitory effect of A beta on K(+)-evoked ACh release also was observed in the frontal cortex but not in the striatum. Taken together, our results demonstrate that APP-derived A beta-related peptides can regulate the release of ACh potently by acting on cholinergic terminals. Additionally, the evidence that selected cholinergic neuronal populations are sensitive to A beta suggests a potential mechanistic link between the deposition of A beta and the preferential vulnerability of certain cholinergic projections in AD.

High affinity choline transporter status in Alzheimer's disease tissue from rapid autopsy.

The degeneration of nucleus basalis cholinergic neurons in Alzheimers disease (AD) has led to therapies that attempt to increase the synaptic availability of acetylcholine in the remaining cholinergic nerve terminals and to thereby reverse or slow the progressive dementia accompanying the disease process. The inadequacy of current choline-replacement therapies suggests that utilization of choline may be disordered and the rate-limiting step in acetylcholine synthesis, the high affinity choline transporter, may be involved. An adequate test of this hypothesis requires the use of fresh, unfrozen tissue, as the transporter activity declines rapidly after death. Using tissue acquired within two hours of death, the activity of the high affinity choline transporter was shown to be increased in cortical brain regions from AD patients compared to non-AD controls. Further studies using frozen tissues with similar short postmortem acquisition times, revealed the expression of the high affinity uptake transporter to be increased in AD cortex as well. When the ratio of regional uptake activity or expression to the regional level of choline acetyltransferase was calculated, the increase in choline transporter activity and expression was clearly statistically significant. Further statistical significance in the choline transporter activity of the AD group was achieved when the putamen, a region without marked pathology in AD, was used as an internal standard to control for agonal state differences in the individual patients contributing tissue to this study. These increases in choline transporter expression and activity in AD indicate disordered regulation of this rate-limiting component of acetylcholine synthesis above and beyond that required to compensate for the reduced cholinergic synaptic availability in AD.

The effect of ovariectomy and estradiol replacement on brain-derived neurotrophic factor messenger ribonucleic acid expression in cortical and hippocampal brain regions of female Sprague-Dawley rats

Alzheimer's disease (AD) is a progressive neurodegenerative disorder whose etiology is presently unknown. Probably the most consistent and widespread deficit seen in this syndrome is that of the basal forebrain cholinergic system. We have previously demonstrated that estradiol (E2) modulates the function of these neurons and plays a role in their maintenance by preventing the ovariectomy-induced decrease in choline acetyltransferase activity. It has been postulated that the lack of neurotrophic support may contribute at least in part to degeneration of cholinergic neurons in AD. As such, it is hypothesized that E2 may affect cholinergic function by modulating the levels of certain neurotrophic factors. We have shown that 3 months after ovariectomy (OVX) there was a significant reduction in NGF messenger RNA (mRNA) levels. In the present study, we extended the hypothesis that E2 may serve a neurotrophomodulatory role by assessing the effect of OVX and E2 replacement on brain-derived nerve factor (BDNF) mRNA levels using in situ hybridization. BDNF mRNA levels were quantified in three groups of animals: ovary-intact animals, 28-week ovariectomized (OVX) animals, and E2-replaced OVX animals. Twenty-eight weeks after OVX, there were significant reductions in two of the three cerebral cortical regions analyzed [frontal (35%) and temporal (39%) cortexes], but E2 replacement was without effect. Twenty-eight weeks after OVX, there were also reductions in BDNF mRNA in all subregions of the hippocampus except CA1 (CA2 by 38%, CA3 by 44%, CA4 by 39%, and dentate gyrus by 37%), whereas E2 replacement was effective in elevating BDNF mRNA levels in the CA3, CA4, and dentate gyrus subregions. Collectively, the data demonstrate that E2 deprivation leads to a reduction in BDNF mRNA. Further, at the time point studied, E2 replacement is more effective in maintaining BDNF mRNA in the hippocampus than in the cortex, suggesting a regional difference in the ovarian steroid requirement for expression of BDNF.