Correlation of choline acetyltransferase activity between the nucleus basalis of Meynert and the cerebral cortex

Choline acetyltransferase (ChAT) activities were measured in the cerebral cortex and the nucleus basalis of Meynert (nbM) of post-mortem human brains from 8 cases with Alzheimer type dementia (ATD) and 5 age-matched control subjects. The lowest ChAT activity was detected in the temporal cortex (Brodmann's area 22) and the nbM in ATD. A significant correlation was found between ChAT activities in the nbM and those in Brodmann's areas 4, 7, 10, 17 and 22. Present results provide evidence of a cholinergic projection from the nbM to the cerebral cortex observed by retrograde or anterograde degeneration studies in animals.     

Nucleus basalis neuronal loss, neuritic plaques and choline acetyltransferase activity in advanced Alzheimer's disease

All our advanced, severe cases of Alzheimer's disease have dramatic cholinergic cell losses in the nucleus basalis of Meynert even after correction for cell or nucleoli shrinkage. There is a good correlation between choline acetyltransferase activity and "healthy" cell number in the nucleus basalis of Meynert. Half of the Alzheimer disease cases have markedly reduced cortical choline acetyltransferase activity in spite of preserved nucleus basalis of Meynert choline acetyltransferase activity, suggesting a deficiency of cortical origin and/or of axonal transport in Alzheimer disease. The relationship between cell loss in the various sub-divisions of the nucleus basalis of Meynert and plaque counts in corresponding and non-corresponding projection areas of the cortex has also been examined. Globally, this relation appears more obvious when cell loss in a sub-division of the nucleus basalis of Meynert is compared to plaque counts in its cortical projection area. However, the relation is discontinuous with few or no data to document the intermediary stages of the process, probably reflecting the severity of our Alzheimer disease cases.     

Regional decreases of cortical choline acetyltransferase after lesions of the septal area and in the area of nucleus basalis magnocellularis

Choline acetyltransferase and [³H]choline uptake have been measured in neocortical regions and hippocampus one week after lesions which destroyed the septum bilaterally, and after unilateral lesions in the area of nucleus basalis magnocellularis. Lesions of the septal area, which severely decreased choline acetyltransferase in hippocampus, only moderately decreased choline acetyltransferase in a posterior cortical region and had no effect in frontal and parietal regions. In contrast, lesions which included nucleus basalis magnocellularis decreased choline acetyltransferase markedly in frontal and parietal regions and had less of an effect in the posterior cortical regions. Lesion-induced decreases of [³H]choline uptake paralleled those of choline acetyltransferase. Lesions which included nucleus basalis magnocellularis had no effect on choline acetyltransferase in hippocampus, nucleus accumbens, olfactory tubercle, midbrain or pons-medulla.These results suggest the existence of topographically distinct cholinergic inputs to neocortex. In agreement with previous studies, cholinergic projections from the peripallidal region of nucleus basalis magnocellularis are predominantly to frontal and parietal neocortex. In contrast to previous suggestions, cholinergic projections to neocortex from the septal area are limited to the posterior regions of neocortex.     

Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.     

Nerve growth factor induces a dose-dependent and long-lasting increase of choline acetyltransferase activity in the septal area and hippocampus of uninjured rats

The effect of nerve growth factor (NGF) on the intact septohippocampal cholinergic system of adult rats was studied. Nerve growth factor was continuously infused at different doses (5-100 micrograms) for two weeks into the lateral ventricle of adult rats. Controls received intracerebroventricular infusion of equal amounts of cytochrome c. Nerve growth factor treatment was capable of inducing a dose-dependent increase of choline acetyltransferase activity (ChAT) in septal area and ventral hippocampus. In both areas, the NGF-induced rise of ChAT activity was sustained for at least one week after infusion, then it progressively declined towards control values. By three and five weeks, using NGF at 25 and 100 micrograms respectively, ChAT increase was still significant in both septum and ventral hippocampus. The present findings corroborate a role for NGF in adult septohippocampal cholinergic system and indicate that the "pharmacological" modulation of these neurons by NGF may last several weeks following withdrawal of this trophic factor.     

Basic fibroblast growth factor suppressed the enhancement of choline acetyltransferase activity induced by nerve growth factor

We investigated change of choline acetyltransferase (ChAT) activities in rat embryonic (16 days) septal neuron culture in treatment with basic fibroblast growth factor (bFGF) and nerve growth factor (NGF). Total ChAT activity increased by addition of bFGF, NGF, and bFGF plus NGF with dose-dependent manner. NGF showed much enhancement of specific ChAT activities per mg protein, but bFGF or bFGF plus NGF, respectively showed little or slightly enhanced ChAT activities. In histochemical studies with anti-ChAT antibody staining, cholinergic neurons in NGF-treated culture were stained more strongly than those in other conditioned cultures such as control, bFGF-treated, and bFGF plus NGF-treated cultures. These results suggest that bFGF enhances total ChAT activity but not cellular ChAT activity and further suppresses the enhancement of cellular ChAT activity induced by NGF in septal neuron culture.     

Coexistence of choline acetyltransferase and nerve growth factor receptors in the rat basal forebrain

Choline acetyltransferase (ChAT) and nerve growth factor (NGF) receptors have previously been shown to be expressed in magnocellular forebrain neurones in the rat. We have now examined their colocalization in these neurones. Using monoclonal antibodies raised against ChAT and NGF receptors we demonstrate here a high degree of colocalization.     

Increased expression of estrogen receptor alpha and beta in the nucleus basalis of Meynert in Alzheimer's disease

The human nucleus basalis of Meynert (NBM) is severely affected in Alzheimer's disease (AD). Since estrogens may reduce both the risk and severity of AD, possibly by an action on the cholinergic system, we determined whether estrogen receptors are present in the human NBM and what their changes are in normal aging and in AD. ERalpha was expressed to a higher degree than ERbeta and was localized mainly in the cell nucleus, while ERbeta was mainly confined to the cytoplasm. A significant positive correlation between the percentage of ERalpha nuclear positive neurons and age was found in men but not in women, whereas the proportion of ERbeta cytoplasm positive cells increased during aging in both sexes. In AD the proportion of neurons showing nuclear staining for both ERalpha and beta and cytoplasmic staining for ERbeta was markedly increased. The percentage of ERbeta nuclear positive neurons increased in AD only in women but not in men. The ApoE genotype had no effect on ER expression in the NBM in AD. In conclusion, whereas only minor sex- and age-related changes in both ERs were found in the human NBM, a clear upregulation of ERalpha and beta was observed in AD.     

Nerve growth factor in treatment and pathogenesis of Alzheimer's disease

The etiology of Alzheimer's disease (AD) is still unknown. In addition, this terrible neurodegenerative disease will increase exponentially over the next two decades due to longer lifespan and an aging "baby-boomer" generation. All treatments currently approved for AD have moderate efficacy in slowing the rate of cognitive decline in patients, and no efficacy in halting progression of the disease. Hence, there is an urgent need for new drug targets and delivery methods to slow or reverse the progression of AD. One molecule that has received much attention in its potential therapeutic role in AD is nerve growth factor (NGF). This review will demonstrate data from humans and animals which promote NGF as a potential therapeutic target by (1) outlining the hypothesis behind using NGF for the treatment of AD, (2) reviewing both the normal and AD altered signaling pathways and effects of NGF in the central nervous system (CNS), and (3) examining the results of NGF treatment obtained from animal models of AD and AD patients.     

Long-term actions of vector-derived nerve growth factor or brain-derived neurotrophic factor on choline acetyltransferase and Trk receptor levels in the adult rat basal forebrain

Trophic factor gene therapy may provide a rational treatment strategy for neurodegenerative disease. Recombinant adeno-associated virus vectors, incorporating a neuron-specific promoter driving bicistronic expression of green fluorescent protein and either nerve growth factor or brain-derived neurotrophic factor, transduced 10,000-15,000 neurons in the medial septum for periods of at least six months. Both cholinergic and non-cholinergic neurons expressed green fluorescent protein. Nerve growth factor and brain-derived neurotrophic factor vectors produced up to 50% increases in immunohistochemical detection of the acetylcholine-synthesizing enzyme in septal neurons ipsilateral to the injection. Increased levels of this enzyme, choline acetyltransferase, persisted for six months with the brain-derived neurotrophic factor vector. The nerve growth factor vector increased Trk receptor immunoreactivity in a volume of brain exceeding that of the transduced cells. Counterstaining for the neuronal marker, NeuN, or Nissl substance did not reveal any vector toxicity at any time-point. It therefore appears that the lasting effects of vector-mediated trophic factor gene transfer will offer a new approach for modulating septal cholinergic transmission and Trk receptor activity.     

Expression of acidic fibroblast growth factor (aFGF) and fibroblast growth factor receptor 4 (FGFR4) in breast fibroadenomas

BACKGROUND/AIM: Fibroadenomas are benign tumours composed of both glandular and fibrous tissue. The mechanisms regulating the growth of these tumours and the relation between the stromal and epithelial cells are poorly understood. Acidic fibroblast growth factor (aFGF) is a well known fibroblast activator, which acts through four specific cell surface receptors, among which, fibroblast growth factor receptor 4 (FGFR4) is highly specific. The aim of this study was to evaluate the distribution of aFGF and FGFR4 in specific cell types of fibroadenomas to understand their possible role in the growth of these breast lesions. METHODS: Formalin fixed and paraffin wax embedded tissues from 15 fibroadenomas and peritumoral normal breasts were investigated for the expression of aFGF and FGFR4 using immunohistochemistry. The presence of aFGF mRNA was also investigated using in situ hybridisation. RESULTS: Immunoreactivity for aFGF and FGFR4 was seen in epithelial cells, but it was lacking in myoepithelial cells of both normal tissues and fibroadenomas. Strong FGFR4 immunoreactivity was found in stromal fibroblasts, which were also weakly positive for aFGF. aFGF mRNA was detected in epithelial cells and in some stromal fibroblasts. CONCLUSIONS: These results suggest a paracrine/autocrine modulation of epithelial and stromal cells of fibroadenomas through an aFGF-FGFR4 interaction. This interaction might regulate various cell functions and the growth of fibroadenomas.     

Gender differences in neurotrophin and glutamate receptor expression in cholinergic nucleus basalis neurons during the progression of Alzheimer's disease

The higher incidence rate of Alzheimer's disease (AD) in elderly women indicates that gender plays a role in AD pathogenesis. Evidence from clinical and pharmacologic studies, neuropathological examinations, and models of hormone replacement therapy suggest that cholinergic basal forebrain (CBF) cortical projection neurons within the nucleus basalis (NB), which mediate memory and attention and degenerate in AD, may be preferentially vulnerable in elderly women compared to men. CBF neurons depend on nerve growth factor (NGF) and their cognate receptors (trkA and p75^NTR) for their survival and maintenance. We recently demonstrated a shift in the balance of NGF and its receptors toward cell death mechanisms during the progression of AD. To address whether gender affects NGF signaling system expression within the CBF, we used single cell RNA amplification and custom microarray technologies to compare gene expression profiles of single cholinergic NB neurons in tissue specimens from male and female members of the Religious Orders Study who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild/moderate AD. p75^NTR expression within male cholinergic NB neurons was unchanged across clinical diagnosis, whereas p75^NTR mRNA levels in female NB neurons exhibited a ∼40% reduction in AD compared to NCI. Male AD subjects displayed a ∼45% reduction in trkA mRNA levels within NB neurons compared to NCI and MCI. In contrast, NB neuronal trkA expression in females was reduced ∼50% in both MCI and AD compared to NCI. Reduced trkA mRNA levels were associated with poorer global cognitive performance and higher Braak scores in the female subjects. In addition, we found a female-selective reduction in GluR2 AMPA glutamate receptor subunit expression in NB neurons in AD. These data suggest that cholinergic NB neurons in females may be at greater risk for degeneration during the progression of AD and support the concept of gender-specific therapeutic interventions during the preclinical stages of the disease.     

Genetic variation in the choline acetyltransferase (CHAT) gene may be associated with the risk of Alzheimer's disease

Several independent linkage studies have mapped a broad susceptibility region for Alzheimer's disease (AD) on the long arm of chromosome 10. There are several biological candidate genes in this region, including choline acetyltransferase (CHAT). A number of studies have examined the role of CHAT genetic variants with AD risk and age-at-onset (AAO), but the results are equivocal. We examined the association of three Single Nucleotide Polymorphisms (SNPs) in the CHAT gene in 1001 white sporadic late-onset AD (LOAD) cases and 708 white controls. We also examined the role of these three SNP with quantitative traits of AD including AAO, disease duration, and Mini-Mental State Examination (MMSE) score. We observed both allelic and genotypic associations of the intron 9 SNP with AD risk in the total sample (p = 0.029 for genotype and p = 0.028 for allele frequency differences) as well as among non-APOE*4 carriers (p = 0.007 for genotype and p = 0.006 for allele frequency differences). Three-site haplotype analysis confirmed that haplotypes determined by the intron 9 SNP were associated with either risk (p = 0.0009) or protective (p = 0.0082) effects among non-APOE*4 carriers. The three CHAT SNPs also showed a modest association with MMSE score. Our data suggest that genetic variation in the CHAT gene may be associated with AD risk and quantitative traits related to AD.     

Cell loss and shrinkage in the nucleus basalis Meynert complex in Alzheimer's disease

Marked neuron loss in the nucleus basalis of Meynert complex (NBMC) in Alzheimer's disease has repeatedly been reported in the literature. However, most of these studies quantitated only magnocellular, hyperchromatic (putative cholinergic) neurons of just a small part of the NBMC, and counts were expressed as numerical density. Applying a 3-dimensional-sampling design throughout the entire rostrocaudal extent of the NBMC and sampling neurons regardless of their size and staining characteristics, an overall neuron loss of only 15.5% was demonstrated for the whole NBMC. Neuron loss varied from 0% rostrally in the NBMC up to 36% in the most caudal part of the nucleus basalis of Meynert. Moreover, a significant increase in the number of small-sized neurons and a significant decrease in the number of large, putative cholinergic neurons could be detected, suggesting that apart from neuron loss neuron shrinkage appears to be another characteristic neuropathological feature of this degenerating cholinergic NBMC system. Preservation of these magnocellular cholinergic neurons in shrunken form renders it likely that cholinergic dysfunction, characteristic of Alzheimer's disease, may be responsive to neurotrophic influences.     

Genetic polymorphisms of nerve growth factor receptor (NGFR) and the risk of Alzheimer's disease

Background

Loss of basal forebrain cholinergic neurons is attributable to the proapoptotic signaling induced by nerve growth factor receptor (NGFR) and may link to Alzheimer's disease (AD) risk. Only one study has investigated the association between NGFR polymorphisms and the risk of AD in an Italian population. Type 2 diabetes mellitus (DM) may modify this association based on previous animal and epidemiologic studies.

Methods

This was a case-control study in a Chinese population. A total of 264 AD patients were recruited from three teaching hospitals between 2007 to 2010; 389 controls were recruited from elderly health checkup and volunteers of the hospital during the same period of time. Five common (frequency??5%) haplotype-tagging single nucleotide polymorphisms (htSNPs) were selected from NGFR to test the association between NGFR htSNPs and the risk of AD.

Results

Variant NGFR rs734194 was significantly associated with a decreased risk of AD [GG vs. TT copies: adjusted odds ratio (OR) = 0.43, 95% confidence interval (CI) = 0.20-0.95]. Seven common haplotypes were identified. Minor haplotype GCGCG was significantly associated with a decreased risk of AD (2 vs. 0 copies: adjusted OR = 0.39, 95% CI = 0.17-0.91). Type 2 DM significantly modified the association between rs2072446, rs741072, and haplotype GCTTG and GTTCG on the risk of AD among ApoE ??4 non-carriers (P _interaction < 0.05).

Conclusion

Inherited polymorphisms of NGFR were associated with the risk of AD; results were not significant after correction for multiple tests. This association was further modified by the status of type 2 DM.     

Simultaneous demonstration of choline acetyltransferase and glutamic acid decarboxylase immunoreactivity in the rat interpeduncular nucleus

Double antigen immunohistochemistry was employed to simultaneously examine the distribution of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) immunoreactivity in the rat interpeduncular nucleus (IPN) at the subnuclear and ultrastructural level. ChAT-immunoreactive axons of the fasciculus retroflexus (FR) innervated specific subnuclear divisions of the IPN that possessed GAD-immunoreactive somata and a high density of GAD-immunoreactive axons and terminals. At the ultrastructural level, each of the cholinoceptive subnuclei possessed a characteristic axodendritic synaptic contact. These morphologically distinct synapses were composed of terminals of ChAT-positive FR axons forming asymmetric contacts with dendritic profiles of GAD-positive neurons. An array of symmetric axodendritic contacts with GAD immunoreactivity located pre- and/or postsynaptically was also present in the cholinoceptive subnuclear divisions. The present study provides direct evidence for synaptic interactions between ChAT-immunoreactive FR axons and dendritic processes of GAD-immunoreactive neurons in the rat IPN. Also, GAD-positive terminals arising from possible intrinsic projections contact dendritic profiles of GAD-immunoreactive neurons in receipt of ChAT-positive FR terminals. These results reveal that putative cholinergic afferent inputs and GABAergic intranuclear projections simultaneously innervate a subpopulation of IPN neurons that possess GAD immunoreactivity.     

Rats with nucleus basalis magnocellularis lesions mimic mnemonic symptomatology observed in patients with dementia of the Alzheimer's type

College students, healthy elderly subjects, patients diagnosed with mild or moderate dementia of the Alzheimer's type, as well as rats with small or large lesions of nucleus basalis magnocellularis (NBM) were tested on an order memory task for a 6- or 8-item list of varying spatial locations. Similar patterns of order memory deficits as a function of serial order position were observed in rats with small or large NBM lesions and patients with mild or moderate dementia of the Alzheimer's type. The results provide support for the possibility that rats with NBM lesions might mimic the mnemonic symptomatology of Alzheimer's disease.     

Decrease in choline acetyltransferase activity in the red nucleus of the cat after cerebellar lesion

The use of a microdissection technique and of a very sensitive radioisotopic assay for choline acetyltransferase, the enzyme that synthesizes acetylcholine, showed that the distribution of this enzyme in the feline red nucleus is uneven. Thus, the enzyme content progressively increased in the caudo-rostral direction, the level at the rostral aspect being about twice that at the caudal. Choline acetyltransferase activity in the cat red nucleus was examined after chronic lesions of the cerebellum. Lesions in the area of the dentate and interpositus nuclei were followed by a decrease in enzyme activity in the contralateral red nucleus, whereas no change was seen on the ipsilateral side. Since this enzyme is a very specific marker for cholinergic neurons, the decrease in enzyme activity caused by the lesions suggests the existence of a cholinergic cerebellorubral pathway. This is supported by the finding that the topographical effects on choline acetyltransferase activity in the red nucleus produced by selective lesions of either the dentate or the interpositus nucleus reflected the arrangement of the cerebellorubral pathways.Choline acetyltransferase activity also decreased in the nucleus ventralis lateralis and the nucleus ventralis anterior of the thalamus contralateral to the lesions. Since cerebello-rubral fibres are thought to be collaterals of cerebello-thalamic axons, at least in the case of these originating in the interpositus nucleus, this would indicate the presence of cholinergic fibres in the brachium conjunctivum (or superior cerebellar peduncle).The results are not in complete agreement with some of the previously reported pharmacological studies; this is discussed.     

Correlation between cortical plaque count and neuronal loss in the nucleus basalis in Alzheimer's disease

In order to substantiate the hypothesis of a cholinergic matrix for neuritic plaque formation in Alzheimer's disease (AD), the relationship between the loss of cholinergic neurons in the basal forebrain and formation of neuritic plaques in the cerebral cortex was studied in 5 cases of neuropathologically verified AD. Quantitative evaluation of the number of neuritic plaques in the cerebral cortex as calculated from 6 areas showed a strong correlation with the loss of neurons in the nucleus basalis of Meynert (NbM). This finding indicates that changes in cortical cholinergic innervation which arise from the neurons of the NbM are an important feature in the pathogenesis of neuritic plaques.