TrkA gene ablation in basal forebrain results in dysfunction of the cholinergic circuitry

Dysfunction of basal forebrain cholinergic neurons (BFCNs) is an early pathological hallmark of Alzheimer's disease (AD). Numerous studies have indicated that nerve growth factor (NGF) supports survival and phenotypic differentiation of BFCNs. Consistent with a potential link to AD pathogenesis, TrkA, a NGF receptor, is expressed in cholinergic forebrain neuronal populations including those in BF and striatum, and is markedly reduced in individuals with mild cognitive impairment (MCI) without dementia and early-stage AD. To investigate the role of TrkA in the development, connectivity, and function of the BF cholinergic system and its contribution to AD pathology, we have generated a forebrain-specific conditional TrkA knock-out mouse line. Our findings show a key role for TrkA signaling in establishing the BF cholinergic circuitry through the ERK pathway, and demonstrate that the normal developmental increase of choline acetyltransferase expression becomes critically dependent on TrkA signaling before neuronal connections are established. Moreover, the anatomical and physiological deficits caused by lack of TrkA signaling in BFCNs have selective impact on cognitive activity. These data demonstrate that TrkA loss results in cholinergic BF dysfunction and cognitive decline that is reminiscent of MCI and early AD.     

Loss of nerve growth factor receptor-containing neurons in Alzheimer's disease: a quantitative analysis across subregions of the basal forebrain

Magnocellular neurons comprising the Ch1-Ch4 regions of the basal forebrain provide topographic cholinergic innervation to the cerebral cortex, thalamus, and basolateral nucleus of the amygdala. Most quantitative studies analyzing the status of these neurons in Alzheimer's disease (AD) have employed Nissl-stained preparations. These studies principally analyzed large neurons of a prespecified cell diameter. Since basal forebrain neurons atrophy in Alzheimer's disease, an immunocytochemical marker for these neurons would appear to be a better alternative for determining whether there is regionally specific degeneration of cholinergic neurons across subregions of the basal forebrain. Brain sections from seven AD and five aged-matched control patients were immunocytochemically stained with a monoclonal antibody raised against the receptor for nerve growth factor (NGF), a probe which has previously been demonstrated to extensively and exclusively colocalize with cholinergic basal forebrain neurons in humans (17, 25, 35). NGF receptor-immunoreactive neurons within the hippocampal projecting nuclei of the medial septum (Ch1) and vertical limb of the diagonal band (Ch2) were minimally affected in AD as compared to control cases. In contrast, the Ch4 region demonstrated a significant loss of NGF receptor-immunoreactive neurons in AD that inversely correlated (-0.786) with the duration of the disease process. All four subregions of Ch4 were affected in the AD cases with the anterolateral (76.4%), intermediate (62.1%) and posterior divisions (76.5%) demonstrating the greatest reduction in NGF receptor-immunoreactive neurons. Nissl-counterstained sections failed to reveal magnocellular neurons which were not immunoreactive for the NGF receptor, suggesting that reductions in immunocytochemically stained neurons reflects neuron loss and not the failure of viable neurons to synthesize NGF receptors. These data indicate that cholinergic basal forebrain neurons which project to the amygdala, as well as to the temporal, frontobasal, and frontodorsal cortices, are most affected in AD.     

Impact of the NGF maturation and degradation pathway on the cortical cholinergic system phenotype

Cortical cholinergic atrophy plays a significant role in the cognitive loss seen with aging and in Alzheimer's disease (AD), but the mechanisms leading to it remain unresolved. Nerve growth factor (NGF) is the neurotrophin responsible for the phenotypic maintenance of basal forebrain cholinergic neurons in the mature and fully differentiated CNS. In consequence, its implication in cholinergic atrophy has been suspected; however, no mechanistic explanation has been provided. We have previously shown that the precursor of NGF (proNGF) is cleaved extracellularly by plasmin to form mature NGF (mNGF) and that mNGF is degraded by matrix metalloproteinase 9. Using cognitive-behavioral tests, Western blotting, and confocal and electron microscopy, this study demonstrates that a pharmacologically induced chronic failure in extracellular NGF maturation leads to a reduction in mNGF levels, proNGF accumulation, cholinergic degeneration, and cognitive impairment in rats. It also shows that inhibiting NGF degradation increases endogenous levels of the mature neurotrophin and increases the density of cortical cholinergic boutons. Together, the data point to a mechanism explaining cholinergic loss in neurodegenerative conditions such as AD and provide a potential therapeutic target for the protection or restoration of this CNS transmitter system in aging and AD.     

Acute cholinergic rescue of synaptic plasticity in the neurodegenerating cortex of anti-nerve-growth-factor mice

Deficits in cholinergic systems innervating cerebral cortex are associated with cognitive impairment during senescence and in age-related neurodegenerative pathologies. However, little is known about the role of cholinergic pathways in modulating cortical plasticity. Basal forebrain cholinergic neurons are a major target for nerve-growth factor (NGF). In order to investigate the relationship between cholinergic innervation and cortical synaptic plasticity, we exploited a transgenic mouse model in which the activity of NGF in the adult nervous system is neutralized by the expression of blocking antibodies to NGF itself (anti-NGF mice) [Ruberti, F. et al. (2000). J. Neurosci. 20, 2589-2601]. In 6-month-old anti-NGF mice, we show that the reduction in cholinergic innervation of the cortex is associated with different forms of synaptic plasticity impairment. A local, acute increase in the availability of acetylcholine rescues these synaptic plasticity deficits, thus indicating that a cholinergic system mediates the impairment of cortical plasticity at this early stage of the neurodegenerative process triggered by NGF neutralization. Our results represent an important step in unveiling the pivotal role of cholinergic transmission in modulating adult cortical plasticity.     

Cholinergic mechanisms in learning, memory and dementia: a review of recent evidence.

The discovery in the late 1970s that cholinergic neurons in the basal forebrain degenerate in Alzheimer's disease (AD) greatly accelerated research on the role of cholinergic mechanisms in learning and memory. As is often the case in science, the early enthusiasm for the cholinergic hypothesis has been tempered by the results of subsequent research. Although there is substantial pharmacological evidence that unspecified cholinergic systems in the brain play important roles in some forms of learning and memory, recent findings in humans indicate that antimuscarinic drugs do not model the deficits seen in AD. In addition, the goal of elucidating the functions of these basal forebrain neurons in animals has proved to be difficult and is yet to be achieved. Despite substantial effort, therefore, the cognitive and behavioral consequences of cholinergic pathology in AD remain unknown. Under these circumstances, attempts to develop cholinergic pharmacotherapies for these deficits in AD are based on questionable assumptions.     

Cholinergic system during the progression of Alzheimer's disease: therapeutic implications

Alzheimer's disease (AD) is characterized by a progressive phenotypic downregulation of markers within cholinergic basal forebrain (CBF) neurons, frank CBF cell loss and reduced cortical choline acetyltransferase activity associated with cognitive decline. Delaying CBF neurodegeneration or minimizing its consequences is the mechanism of action for most currently available drug treatments for cognitive dysfunction in AD. Growing evidence suggests that imbalances in the expression of NGF, its precursor proNGF and the high (TrkA) and low (p75(NTR)) affinity NGF receptors are crucial factors underlying CBF dysfunction in AD. Drugs that maintain a homeostatic balance between TrkA and p75(NTR) may slow the onset of AD. A NGF gene therapy trial reduced cognitive decline and stimulated cholinergic fiber growth in humans with mild AD. Drugs treating the multiple pathologies and clinical symptoms in AD (e.g., M1 cholinoceptor and/or galaninergic drugs) should be considered for a more comprehensive treatment approach for cholinergic dysfunction.     

Nerve growth factor infusion in the primate brain reduces lesion-induced cholinergic neuronal degeneration

NGF is a protein that promotes survival, differentiation, and process extension of selected neuronal populations during development and, in some cases, in the mature organism. Previous lesion and aging studies in the rat have shown that intracerebroventricular NGF infusions can prevent degenerative changes in basal forebrain cholinergic neurons. We sought to determine whether salutory effects of NGF occur in the primate brain. Cholinergic fibers of the septohippocampal projection in the primate were surgically transected, followed by infusion of either a vehicle or an NGF solution into the ventricular system for a 4-week period. Quantification of cholinergic neurons in the medial septal nucleus at the end of the infusion period demonstrated that only 45 +/- 5% of cholinergic neurons could be identified after fornix lesions in vehicle-infused animals, whereas 80 +/- 6% of neurons were visible in NGF-treated animals. Thus, NGF substantially reduced lesion-induced cholinergic neuronal degeneration in the adult primate brain. This finding may be relevant to the hypothesis that NGF has potential use as a cholinergic "neurotrophic-factor therapy," given that loss of basal forebrain cholinergic neurons is common in Alzheimer's disease.     

Nerve growth factor receptor-mediated transport from CSF labels cholinergic neurons: direct demonstration by a double-labeling study

It has recently been shown that, following intraventricular administration, a monoclonal antibody directed against rat nerve growth factor (NGF) receptor is specifically accumulated bilaterally by numerous neurons of the basal forebrain. The labeled neurons have a morphology and topography which are characteristic of the magnocellular cholinergic basal forebrain (CBF) system. This is also consistent with the evidence that CBF neurons have NGF receptors and respond to NGF in a variety of experimental situations. In the present report, a double-labeling technique is used to directly demonstrate that choline acetyltransferase-containing (and therefore cholinergic) neurons do in fact accumulate the anti-NGF receptor antibody from CSF.     

APP, NGF & the 'Sunday-driver' in a Trolley on the Road

Alzheimer's disease (AD) is one of the most common and challenging neurodegenerative diseases in humans and is characterized by: progressive impairment in cognitive function, degeneration of cholinergic neurons of the basal forebrain (CBF), neurofibrillary tangles and amyloid beta-peptide (Abeta) depositions. The amyloid precursor protein (APP) is a transmembrane protein of which abnormal processing produces Abeta that is associated with the pathogenesis of AD. Neurotrophic factors have attracted much attention for their potential as a remedy for neurological disorders. In this regard, nerve growth factor (NGF) has generated a great interest as a potential target for the treatment of AD. This interest is based on the observation that CBF neurons, which provide the major source of cholinergic innervation to the cerebral cortex and hippocampus, undergo selective and severe degeneration in advanced AD and that the survival of CBF neurons depends upon NGF and its receptors, namely, trkA and p75NTR. This review focuses on recent findings about APP, NGF and their potential signaling-connections to the protein encoded by the 'Sunday-driver' (SYD) gene.     

Long-term reversal of cholinergic neuronal decline in aged non-human primates by lentiviral NGF gene delivery

Spontaneous atrophy of basal forebrain cholinergic neurons occurs with aging in the non-human primate brain. Short-term reversal of this atrophy has been reported following ex vivo nerve growth factor (NGF) gene delivery, but long-term effects of in vivo NGF gene delivery in the aged primate brain have not to date been examined. We tested the hypothesis that long-term lentiviral NGF intraparenchymal gene delivery would reverse age-related cholinergic decline, without induction of adverse effects previously observed following sustained intracerebroventricular growth factor protein exposure. Three aged rhesus monkeys underwent intraparenchymal lentiviral NGF gene delivery to the cholinergic basal forebrain. 1 year later, cholinergic neuronal numbers were quantified stereologically and compared to findings in four controls, non-treated aged monkeys and four young adult monkeys. Safety was assessed on several variables related to growth factor exposure. We now report that lentiviral gene delivery of NGF to the aged primate basal forebrain sustains gene expression for at least 1 year, and significantly restores cholinergic neuronal markers to levels of young monkeys. Aging resulted in a significant 17% reduction (p < 0.05) in the number of neurons labeled for the cholinergic marker p75 among basal forebrain neurons. Lentiviral NGF gene delivery induced significant (p < 0.05) and nearly complete recovery of p75-labeled neuronal numbers in aged subjects to levels observed in young monkeys. Similarly, the size of cholinergic neurons in aged monkeys was significantly reduced by 16% compared to young subjects (p < 0.05), and lentiviral NGF delivery to aged subjects induced complete recovery of neuronal size. Intraparenchymal NGF gene delivery over a one-year period did not result in systemic leakage of NGF, activation of inflammatory markers in the brain, pain, weight loss, Schwann cell migration, or formation of anti-NGF antibodies. These findings indicate that extended trophic support to neurons in the non-human primate brain reverses age-related neuronal atrophy. These findings also support the safety and feasibility of lentiviral NGF gene transfer for potential testing in human clinical trials to protect degenerating cholinergic neurons in Alzheimer's disease.     

Nerve growth factor and Alzheimer's disease

Alzheimer's disease is associated with a pronounced loss of the cholinergic neurons that form the ascending cholinergic projections of the basal forebrain. Even though the disease is also characterized by changes in other neuronal systems and by a high frequency of neuronal plaques and tangles, the cholinergic deficit seems to be a principal element responsible for the memory loss typical of Alzheimer's disease. This review summarizes findings in experimental animals which indicate that nerve growth factor (NGF), a well-characterized protein, acts as a neurotrophic factor for cholinergic neurons of the basal forebrain. NGF is present in the target areas of these cholinergic neurons and affects their survival, fiber growth, and expression of transmitter-specific enzymes. Furthermore, NGF is able to prevent the degeneration of cholinergic neurons in adult rats with experimental lesions mimicking the cholinergic deficit in Alzheimer's disease. These findings suggest that increasing the availability of NGF to human cholinergic cells might promote their survival in certain disease processes. Additional steps are discussed for establishing the possible involvement of NGF in the pathogenesis of Alzheimer's disease and the development of an effective therapy.     

Time window in cholinomimetic ability to rescue long-term potentiation in neurodegenerating anti-nerve growth factor mice

A deficit in cortical cholinergic synaptic transmission is a common feature of cognitive and behavioral impairment observed in neurodegenerative pathologies. AD11 transgenic mice producing blocking antibodies against Nerve Growth Factor (NGF) are characterized by a progressive neurodegenerative phenotype defined by the deposition of amyloid peptide, intracellular neurofibrillary tangles and by a marked cholinergic depletion. We exploited AD11 mice to develop a functional assay to investigate the impact of cholinergic deficit on cortical synaptic plasticity impairment at different neurodegenerative stages. In particular, we investigated the time course of long-term potentiation (LTP) impairment in neocortex of AD11 mice and potential rescue by acute pharmacological treatment with Acetylcholine (ACh) or the cholinergic agonist Galantamine (GAL). We showed that LTP starts being absent in AD11 mice at 2 months, an age corresponding to early neurodegenerative stage characterized by the first observed decrease in number of basal forebrain cholinergic neurons (BFCNs) without overt cortical neurodegeneration. We demonstrated that acute ACh or GAL treatment fully reverts LTP impairment in 2 month old AD11 mice. In contrast, cholinergic treatment failed to recover synaptic plasticity deficit in aged (9-10 months) AD11 mice characterized by a severe cortical neurodegeneration.     

NGF Deprivation of Adult Rat Brain Results in Cholinergic Hypofunction and Selective Impairments in Spatial Learning

Cholinergic hypofunction has often been correlated with a variety of behavioural impairments. In the present study, adult Wistar rats were intraventricularly infused with antibodies to nerve growth factor (anti-NGF) to examine the effects on cholinergic neurons of the basal forebrain, and on behavioural performance. Immunocytochemical techniques indicated that chronically infused anti-NGF penetrates into the basal forebrain, cortex, striatum, corpus callosum and hippocampus, confirming previous findings after a single injection. Treatment with anti-NGF for 1 or 2 weeks resulted in a significant decrease of 27-33% in density of choline acetyltransferase immunostaining of the cholinergic cell bodies in the medial septum and vertical diagonal band, and a 26% reduction in choline acetyltransferase enzyme activity in the septal area. An array of spatial learning Morris water maze tasks was used to distinguish between acquisition skills and the flexible use of learned information in novel tests. Rats subjected to the spatial learning paradigm received anti-NGF infusion for 2 weeks prior to and for another 2 weeks during the behavioural testing. The anti-NGF-treated animals were found to be no different from those receiving control serum in the Morris water maze acquisition task, either in the latency to find the platform or in the time spent searching in the training quadrant when the platform was removed. However, in consecutive extinction trials, anti-NGF rats continued to search in the empty training quadrant, suggesting the occurrence of perseveration; control rats expanded their search over other areas of the pool. This inflexibility of the anti-NGF rats was also evident from their difficulty in learning to find a relocated platform in the reversal task. Finally, the anti-NGF-treated animals demonstrated hyperactivity in the open field, resembling in this respect the behaviour of animals after septal and fimbria fornix lesions, and during pharmacological cholinergic blockade. While these data support a role for NGF in cholinergic function and spatial learning behaviour, they indicate that deficits in the latter, e.g. those demonstrated in impaired aged rats, may not be attributable selectively to deficits in the function of NGF-sensitive cholinergic neurons of the basal forebrain.     

Neurofibrillary degeneration of cholinergic and noncholinergic neurons of the basal forebrain in Alzheimer's disease

Two principal features of Alzheimer's disease (AD) are (1) the occurrence of neurofibrillary tangles (NFTs) and senile plaques, and (2) the loss of cortical cholinergic activity because of dysfunction of neurons in the basal forebrain cholinergic system. The relationship of these two abnormalities is an unresolved issue in the pathology of AD. We used polyclonal antibodies specific for paired helical filaments (PHFs), combined with acetylcholinesterase (AChE) histochemistry, to assess the cytoskeletal changes of cholinergic and noncholinergic neurons in the basal forebrain in AD. In both sporadic and familial AD, the nucleus basalis of Meynert (nbM) showed a marked decrease in AChE-positive (AChE+) perikarya and abundant immunoreactive NFTs. In double-labeling studies of the nbM, PHF reactivity was found both in surviving AChE+ neurons and in many AChE- NFTs that were not associated with microscopically recognizable cell structures. Some surviving AChE+ perikarya did not contain NFTs. Numerous NFTs and senile plaques were identified by PHF immunoreactivity in other basal forebrain areas, including subnuclei of the amygdala that showed low or absent AChE activity. We conclude that the dysfunction and death of cholinergic neurons in the nbM is associated with extensive NFT formation, including apparently residual NFTs in loci where nbM neurons once existed; and many noncholinergic neurons and neurites in the basal forebrain show NFT and senile plaque formation. The cytopathology of AD involves neurons of varying transmitter specificities, including cholinergic neurons in the basal forebrain.     

Loss of medial septum cholinergic neurons in THY-Tau22 mouse model: what links with tau pathology?

Alzheimer's disease (AD) is a neurodegenerative disorder histologically defined by the cerebral accumulation of amyloid deposits and neurofibrillary tangles composed of hyperphosphorylated tau proteins. Loss of basal forebrain cholinergic neurons is another hallmark of the disease thought to contribute to the cognitive dysfunctions. To this date, the mechanisms underlying cholinergic neurons degeneration remain uncertain. The present study aimed to investigate the relationship between neurofibrillary degeneration and cholinergic defects in AD using THY-Tau22 transgenic mouse model exhibiting a major hippocampal AD-like tau pathology and hyperphosphorylated tau species in the septohippocampal pathway. Here, we report that at a time THY-Tau22 mice display strong reference memory alterations, the retrograde transport of fluorogold through the septohippocampal pathway is altered. This impairment is associated with a significant reduction in the number of choline acetyltransferase (ChAT)-immunopositive cholinergic neurons in the medial septum. Analysis of nerve growth factor (NGF) levels supports an accumulation of the mature neurotrophin in the hippocampus of THY-Tau22 mice, consistent with a decrease of its uptake or retrograde transport by cholinergic terminals. Finally, our data strongly support that tau pathology could be instrumental in the cholinergic neuronal loss observed in AD.     

The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease

Nerve growth factor (NGF) is important for regulation, differentiation, and survival of peripheral and central nervous system neurons, including basal forebrain cholinergic neurons (BFCN) which degenerate in Alzheimer's disease (AD). Mature NGF protein is processed from a larger precursor, proNGF. We demonstrate that proNGF is the predominant form of NGF in mouse, rat, and human brain tissue, whereas little or no mature NGF is detected. Previous reports showed NGF protein, measured by ELISA, is increased in AD BFCN target regions such as hippocampus and cortex. Using Western blotting, we demonstrate a twofold increase in proNGF in AD parietal cortex compared to controls, indicating that it is this precursor form, proNGF, that accumulates in AD. This increase may reflect either a role for biologically active proNGF or posttranslational disturbances in NGF biosynthesis that decrease the processing of proNGF to mature NGF in AD.     

Reversible schwann cell hyperplasia and sprouting of sensory and sympathetic neurites after intraventricular administration of nerve growth factor

Substantial dysfunction and loss of cholinergic neurons occur in Alzheimer's disease (AD). Nerve growth factor (NGF) is a potent neurotrophic factor for cholinergic basal forebrain neurons, and the use of NGF to stimulate residual dysfunctional cells in AD is being considered. To define the effects of NGF on other cell populations in the brain, NGF was continuously infused into the lateral ventricle of rats for 7 weeks. At the end of treatment, Schwann cell hyperplasia and abundant sensory and sympathetic neurite sprouting were observed in the subpial region of the medulla oblongata and the spinal cord. Following withdrawal of NGF, the Schwann cell hyperplasia and sprouting of sensory and sympathetic neurites disappeared completely. These findings suggest that better temporal and spatial delivery systems for NGF must be explored to limit potential undesirable side effects while maintaining the survival and function of diseased basal forebrain cholinergic neurons.     

The cholinergic system in aging and neuronal degeneration

The basal forebrain cholinergic complex comprising medial septum, horizontal and vertical diagonal band of Broca, and nucleus basalis of Meynert provides the mayor cholinergic projections to the cerebral cortex and hippocampus. The cholinergic neurons of this complex have been assumed to undergo moderate degenerative changes during aging, resulting in cholinergic hypofunction that has been related to the progressing memory deficits with aging.However, the previous view of significant cholinergic cell loss during aging has been challenged. Neuronal cell loss was found predominantly in pathological aging, such as Alzheimer's disease, while normal aging is accompanied by a gradual loss of cholinergic function caused by dendritic, synaptic, and axonal degeneration as well as a decrease in trophic support. As a consequence, decrements in gene expression, impairments in intracellular signaling, and cytoskeletal transport may mediate cholinergic cell atrophy finally leading to the known age-related functional decline in the brain including aging-associated cognitive impairments.However, in pathological situations associated with cognitive deficits, such as Parkinsons's disease, Down-syndrome, progressive supranuclear palsy, Jakob-Creutzfeld disease, Korsakoff's syndrome, traumatic brain injury, significant degenerations of basal forebrain cholinergic cells have been observed. In presenile (early onset), and in the advanced stages of late-onset Alzheimer's disease (AD), a severe loss of cortical cholinergic innervation has extensively been documented. In contrast, in patients with mild cognitive impairment (MCI, a prodromal stage of AD), and early forms of AD, apparently no cholinergic neurodegeneration but a loss of cholinergic function occurs. In particular imbalances in the expression of NGF, its precursor proNGF, the high and low NGF receptors, trkA and p75NTR, respectively, changes in acetylcholine release, high-affinity choline uptake, as well as alterations in muscarinic and nicotinic acetylcholine receptor expression may contribute to the cholinergic dysfunction. These observations support the suggestion of a key role of the cholinergic system in the functional processes that lead to AD. Malfunction of the cholinergic system may be tackled pharmacologically by intervening in cholinergic as well as neurotrophic signaling cascades that have been shown to ameliorate the cholinergic deficit at early stages of the disease, and slow-down the progression. However, in contrast to many other, dementing disorders, in AD the cholinergic dysfunctions are accompanied by the occurrence of two major histopathological hallmarks such as β-amyloid plaques and neurofibrillary tangles, provoking the question whether they play a particular role in inducing or mediating cholinergic dysfunction in AD. Indeed, there is abundant evidence that β-amyloid may trigger cholinergic dysfunction through action on α7 nicotinic acetylcholine receptors, affecting NGF signaling, mediating tau phosphorylation, interacting with acetylcholinesterase, and specifically affecting the proteome in cholinergic neurons. Therefore, an early onset of an anti β-amyloid strategy may additionally be potential in preventing aging-associated cholinergic deficits and cognitive impairments.     

β/A4-Amyloid increases nerve growth factor production in rat primary hippocampal astrocyte cultures

Nerve growth factor (NGF), a member of the neurotrophin family, is an essential mediator of neuronal activity and synaptic plasticity of basal forebrain cholinergic neurons (BFCN). In processes of chronic degeneration of BFCN like in Alzheimer's disease (AD), characterized among others by amyloid containing plaques, NGF has been shown to improve cognitive decline and rescue BFCN but also to reduce survival of hippocampal neurons via p75 neurotrophin receptor (p75). Little is known about the mechanisms of NGF regulation in glial cells under pathological conditions in AD. This study investigates the influence of amyloid administration on the NGF protein secretion in rat primary hippocampal astrocytes. Astrocytes were stimulated with "aged" beta/A4-Amyloid (1-40), and NGF was measured in different fractions, such as supernatant, vesicles, and cytosol fraction. Treatment with amyloid at a final concentration of 10 microM for 72 h led to increased NGF protein levels up to 30-fold increase compared to unstimulated controls. This observation may be an endogenous neuroprotective mechanism possibly contributing to a delay of amyloid-dependent loss of cholinergic neurons or contribute to accelerated neuronal death by activation of p75 within Alzheimer pathology.