Degradation of islet amyloid polypeptide by neprilysin.

AIMS/HYPOTHESIS: A progressive loss of pancreatic beta cell function, a decrease in beta cell mass and accumulation of islet amyloid is characteristic of type 2 diabetes mellitus. The main constituent of islet amyloid is islet amyloid polypeptide (IAPP). In this study, we examined the ability of the peptidase neprilysin to cleave IAPP and prevent human IAPP-induced pancreatic beta cell toxicity. METHODS: Neprilysin and a catalytically compromised neprilysin mutant were tested for their ability to inhibit human IAPP fibrillisation and human IAPP-induced pancreatic beta cell cytotoxicity. Degradation of human IAPP by neprilysin was followed by HPLC, and the degradation products were identified by MS. RESULTS: Neprilysin prevented IAPP fibrillisation by cleaving IAPP at Arg(11)-Leu(12), Leu(12)-Ala(13), Asn(14)-Phe(15), Phe(15)-Leu(16), Asn(22)-Phe(23) and Ala(25)-Ile(26). It also appears to prevent human IAPP fibrillisation through a non-catalytic interaction. Neprilysin protected against beta cell cytotoxicity induced by exogenously added or endogenously produced human IAPP. CONCLUSIONS/INTERPRETATION: The data presented support a potential therapeutic role for neprilysin in preventing type 2 diabetes mellitus. This study supports the hypothesis that extracellular human IAPP contributes to human IAPP-induced beta cell cytotoxicity. Whether human IAPP exerts its cytotoxic effect through a totally extracellular mechanism or through a cellular reuptake mechanism is unclear at this time.

Lentiviral vector neutral endopeptidase gene transfer suppresses prostate cancer tumor growth.

Neprilysin (neutral endopeptidase, NEP) is a cell surface peptidase whose expression is lost in approximately 50% of prostate cancers (PC). NEP normally functions to inactivate peptides such as bombesin and endothelin-1, and potentiates the effects of the PTEN tumor suppressor via a direct protein-protein interaction. NEP loss contributes to PC progression. We investigated the therapeutic efficacy of using a lentiviral vector system to restore NEP expression in PC cells. Third-generation lentiviral vectors encoding wild-type NEP (L-NEP) or green fluorescent protein (L-GFP) were introduced into NEP-deficient 22RV1 PC cells. Cells infected with L-NEP or L-GFP at a multiplicity of infection of 10 demonstrated NEP enzyme activity of 1171.2+/-4.9 and 17.2+/-5.3 pmol/microg/min (P<0.0001), respectively. Cell viability, proliferation and invasion were each significantly inhibited in 22RV1 cells expressing NEP compared with control cells infected with L-GFP (P<0.01). Analysis of known downstream effects of NEP showed NEP-expressing cells exhibiting decreased Akt and focal adhesion kinase phosphorylation and increased PTEN protein expression. Finally, injection of L-NEP into established 22RV1 xenograft tumors significantly inhibited tumor growth (P<0.01). These experiments demonstrate that lentiviral NEP gene transfer is a novel targeted strategy for the treatment of NEP-deficient PC.

Protein kinase A regulates cholinergic gene expression in PC12 cells: REST4 silences the silencing activity of neuron-restrictive silencer factor/REST.

The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A-deficient PC12 mutant, A126.1B2, in which transcription of the gene is reduced. The site of action of protein kinase A was localized to a neuron-restrictive silencer element/repressor element 1 (NRSE/RE-1) sequence within the cholinergic gene. Neuron-restrictive silencer factor (NRSF)/RE-1-silencing transcription factor (REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2 cells. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform REST4 was expressed in PC12 cells but not in A126.1B2. REST4 inhibited binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Coimmunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in A126.1B2 was sufficient to transcriptionally activate the cholinergic gene locus. Thus, in PC12 cells, protein kinase A promotes the production of REST4, which inhibits repression of the cholinergic gene locus by NRSF/REST.

Insulysin hydrolyzes amyloid beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques.

Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.

Increased expression of an endopeptidase (gamma-EGE/IDE) hydrolyzing beta-endorphin during differentiation and maturation of bone marrow macrophages.

The presence and regulated expression of peptidase activity is a powerful mechanism with the potential to terminate or alter receptor recognition, cell membrane signal transduction, and physiological responses of immune cells to exogenous opioid peptides. In this study, the expression of an endopeptidase that hydrolyzes beta-endorphin to gamma-endorphin and other peptide products was investigated during in vitro differentiation and maturation of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) -derived, bone marrow-derived macrophages. In freshly isolated intact isolated mouse bone marrow cells the rate of beta-endorphin hydrolysis is undetectable (<0.1 nmol beta-endorphin hydrolyzed/h/10[6] cells). However, total intracellular beta-endorphin hydrolytic activity was increased significantly to 20.0 +/- 1.7 nmol/h/10(6) cells in the mature mouse macrophages derived in vitro by culture with rGM-CSF. rGM-CSF-derived macrophages expressed significantly higher levels of both protein and mRNA for the major beta-endorphin endopeptidase, gamma-endorphin-generating enzyme/insulin-degrading enzyme (gamma-EGE/IDE). Moreover, this enzymatic activity appears to be responsible for cleavage of exogenous beta-endorphin by intact rGM-CSF-derived macrophages or peritoneal macrophages to generate gamma-endorphin and other peptide products.

Morphologic alterations of cholinergic processes in the neocortex of aged rats.

In the present study we observed enlarged cholinergic processes in the neocortex of aged Fischer 344 rats. These swollen ChAT-positive profiles appeared either as a single axon enlargement or, in many instances, the bulbous processes coalesced to form grape-like clusters of immunoreactivity. The latter structures looked similar to the immunoreactive profiles observed in the cortex of patients with Alzheimer's disease and in the rat septum following fimbria-fornix transection. Together, these data provide evidence that morphologic changes occur within processes of cholinergic neurons in the aged rat. Moreover, the similarity in appearance between the axonal alterations in the aged rat and in patients with Alzheimer's disease suggests a common pathologic process.

Cholinergic fiber aberrations in nucleus basalis lesioned rat and Alzheimer's disease.

Innervation density and morphological aberrations of cholinergic fibers were studied with choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry in 30-35 month-old aged rats and rats with long-term bilateral lesions of the magnocellular basal nucleus (MBN). In addition, AChE histochemistry was performed on human cortical sections derived from autopsy brains of normal aged and Alzheimer's disease (AD) patients. A limited but variable number of morphological alterations were observed in ChAT-immunoreactive fibers in the cortex and the hippocampus of the aged control rats. The aged MBN-lesioned rats displayed a severely reduced number of cholinergic fibers in the denervated areas of the neocortex, whereas the surviving fibers showed a strongly increased number of aberrations. Fiber anomalies were also observed in the cortex of the aged human subjects and Alzheimer patients, the latter showing a higher incidence of such aberrations. Only a part of these distended profiles were seen in close association with senile plaques as detected in the AChE-stained material. These findings suggest that experimental MBN lesions combined with aging share with AD the induction of large quantities of fiber malformations. Implications of possible mechanisms in both conditions are discussed.

Morphologic alterations of choline acetyltransferase-positive neurons in the basal forebrain of aged behaviorally characterized Fisher 344 rats.

We examined Fisher 344 female rats aged 6, 27, and 33 months old. Prior to sacrifice and morphometric analyses of forebrain cholinergic neurons all rats underwent behavioral characterization in a spatial learning task using the Morris water maze. Performance on the spatial task permitted subsequent grouping of the 27- and 33-month-old animals into impaired or nonimpaired groups. Importantly, the percentage of animals that displayed spatial impairments increased sharply with advancing age. Quantitative assessment of the size and density of choline acetyltransferase (ChAT)-positive neurons throughout the basal forebrain revealed a significant enlargement of forebrain cholinergic neurons within 27-month-old nonimpaired rats compared to 6-month-old rats and 27- and 33-month-old impaired animals. This increase in size was most noted in the medial septum and nucleus of the diagonal band. Significant decreases in the density of ChAT-positive neurons was observed only in the nucleus of the diagonal band of 27-month-old impaired rats compared to 6-month-old controls. Although the significance of enlarged forebrain cholinergic neurons is unclear, we discuss the possibility that within aged rodents neuronal swelling is an active event and represents an early manifestation of the aging process and may constitute a restorative and/or compensatory event in that these rats are relatively asymptomatic with respect to their behavioral deficits. In addition, we discuss in some detail various technical and life effect issues which may vary the outcome of investigations of aged rodents.

Comparison of the structure and expression of the human and rat neprilysin (endopeptidase 24.11)-encoding genes.

The existence of a third non-coding exon in the human neprilysin-encoding gene (h-NEP), positionally located as exon 3, has been demonstrated by reverse transcription of RNA from human kidney and lung, coupled with the polymerase chain reaction. Comparison of nucleotide sequences between h-NEP and the rat NEP (r-NEP) genes shows a high degree of sequence conservation within noncoding exons 1 and 2. In contrast, the region of the gene containing exon 3 is highly divergent. Two transcripts derived from exon 2 by alternative splicing, type-2a and type-2b, were demonstrated in human kidney and lung. In contrast, only the type-2b transcript was present in these same tissues in the rat. The type-1 transcript was detected in human kidney, lung and brain, this transcript appearing to be the major species in brain.

Synaptic organization of cholinergic amacrine cells in the rhesus monkey retina.

In the rhesus monkey retina, choline acetyltransferase (ChAT) immunoreactivity has been used to study the localization and synaptic organization of cholinergic neurons by both light and electron microscopy with peroxidase-antiperoxidase immunohistochemistry. ChAT-containing neurons are a type of amacrine cell with 97.5% of their cell bodies localized to the ganglion cell layer and the remainder in the inner nuclear layer. Their processes arborize in a single narrow band in the inner plexiform layer in a plane dividing the outer two-thirds from the inner one-third of this synaptic region. With electron microscopy, ChAT-immunoreactive amacrine cell processes were observed to be primarily postsynaptic to the diffuse invaginating cone bipolar cells and presynaptic to ganglion cells, although they are both post- and presynaptic to immunohistochemically unlabeled amacrine cell profiles and to ChAT-containing amacrine cell processes as well.

Human reticular formation: cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei and some cytochemical comparisons to forebrain cholinergic neurons.

Choline acetyltransferase immunohistochemistry showed that the human rostral brainstem contained cholinergic neurons in the oculomotor, trochlear, and parabigeminal nuclei as well as within the reticular formation. The cholinergic neurons of the reticular formation were the most numerous and formed two intersecting constellations. One of these, designated Ch5, reached its peak density within the compact pedunculopontine nucleus but also extended into the regions through which the superior cerebellar peduncle and central tegmental tract course. The second constellation, designated Ch6, was centered around the laterodorsal tegmental nucleus and spread into the central gray and medial longitudinal fasciculus. There was considerable transmitter-related heterogeneity within the regions containing Ch5 and Ch6. In particular, Ch6 neurons were intermingled with catecholaminergic neurons belonging to the locus coeruleus complex. The lack of confinement within specifiable cytoarchitectonic boundaries and the transmitter heterogeneity justified the transmitter-specific Ch5 and Ch6 nomenclature for these two groups of cholinergic neurons. The cholinergic neurons in the nucleus basalis (Ch4) and those of the Ch5-Ch6 complex were both characterized by perikaryal heteromorphism and isodendritic arborizations. In addition to choline acetyltransferase, the cell bodies in both complexes also had high levels of acetylcholinesterase activity and nonphosphorylated neurofilament protein. However, there were also marked differences in cytochemical signature. For example, the Ch5-Ch6 neurons had high levels of NADPHd activity, whereas Ch4 neurons did not. On the other hand, the Ch4 neurons had high levels of NGF receptor protein, whereas those of Ch5-Ch6 did not. On the basis of animal experiments, it can be assumed that the Ch5 and Ch6 neurons provide the major cholinergic innervation of the human thalamus and that they participate in the neural circuitry of the reticular activating, limbic, and perhaps also extrapyramidal systems.

Nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: colocalization with cholinergic neurons.

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) has been used to map the distribution of NGF receptor-containing profiles within the human basal forebrain of four male and three female elderly patients without neurologic or psychiatric illness. Immunohistochemically processed tissue reveals a continuum of NGF receptor-positive neurons located within the medial septum, vertical and horizontal limb nuclei of the diagonal band, and nucleus basalis. NGF receptor-containing neurons are also found within the bed nucleus of the stria terminalis, the anterior commissure, the internal capsule, and the internal and external medullary laminae of the globus pallidus. Virtually all (greater than 95%) NGF receptor-containing neurons colocalize with the specific cholinergic marker choline acetyltransferase (ChAT) or the nonspecific marker acetylcholinesterase (AChE). Conversely, a few cholinergic perikarya are found which are not NGF receptor positive (and vice versa). These findings demonstrate that human basal forebrain neurons on which NGF receptor immunoreactivity is detected are primarily cholinergic and analogous to the nonhuman primate Ch1-Ch4 subgroups of Mesulam et al. (J. Comp. Neurol., 214:170-197, '83). NGF receptor-containing fiber tracts are observed emanating from the medial septum and vertical limb nucleus of the diagonal band coursing medially within the fornix. Another fascicle originating mainly from the nucleus basalis and travelling within the external capsule enroute to the cortex is observed innervating all cortical layers. Comparison of NGF receptor- and ChAT-containing neurons reveals cholinergic perikarya within the striatal complex, whereas virtually no NGF receptor-containing neurons are found in these structures. An occasional displaced NGF receptor-containing neurons is seen in the ventrolateral portion of the putamen and the white matter underlying the nucleus accumbens. These data are discussed in terms of the relationship of NGF receptor- and ChAT-containing neurons within the basal forebrain and in terms of the possible functional significance of NGF in normal and diseased brain.

Differential cholinergic innervation within functional subdivisions of the human cerebral cortex: a choline acetyltransferase study.

The distribution of cholinergic fibers in the human brain was investigated with choline acetyltransferase immunocytochemistry in 35 cytoarchitectonic subdivisions of the cerebral cortex. All cortical areas and all cell layers contained cholinergic axons. These fibers displayed numerous varicosities and, on occasion, complex preterminal profiles arranged in the form of dense clusters. The density of cholinergic axons tended to be higher in the more superficial layers of the cerebral cortex. Several distinct patterns of lamination were identified. There were also major differences in the overall density of cholinergic axons from one cytoarchitectonic area to another. The cholinergic innervation of primary sensory, unimodal, and heteromodal association areas was lighter than that of paralimbic and limbic areas. Within unimodal association areas, the density of cholinergic axons and varicosities was significantly lower in the upstream (parasensory) sectors than in the downstream sectors. Within paralimbic regions, the non-isocortical sectors had a higher density of cholinergic innervation than the isocortical sectors. The highest density of cholinergic axons was encountered in core limbic structures such as the hippocampus and amygdala. These observations show that the cholinergic innervation of the human cerebral cortex displays regional variations that closely follow the organization of information processing systems.

Cholinergic innervation of the human cerebellum.

Cholinergic innervation of the human cerebellum was investigated immunocytochemically by using a polyclonal rabbit antiserum against choline acetyltransferase. Immunoreactive structures were found throughout the cerebellar cortex but were localized predominantly in the vermis, flocculus, and tonsilla. These included 1) a population of Golgi cells in the granular layer; 2) a subpopulation of mossy fibers and glomerular rosettes; 3) thin, varicose fibers closely associated with the Purkinje cell layer and the molecular layer; and 4) a relatively dense network of fibers and terminals contributing to the glomerular formations in the granular layer. In the cerebellar nuclei, some cells stained positively for choline acetyltransferase, and a terminal field pattern could be detected with a distinct but sparse network of varicose fibers. Acetylcholine appears to be a primary transmitter in the vestibulocerebellar pathways at several levels, which may account for the potent effects of muscarinic antagonists in diminishing vestibular vertigo in humans.

Cholinergic innervation in the human hippocampal formation including the entorhinal cortex.

The cholinergic innervation of the hippocampal formation is thought to play an important role in memory processes, but its organization in humans has not been described in detail. We studied the cholinergic innervation of the human hippocampal formation by means of immunohistochemistry with polyclonal antisera directed against acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and the low-affinity (p75) nerve growth factor receptor (NGFR). The density of ChAT-like immunoreactive (ChAT-li) fibers differed substantially among the various regions, in general paralleling the pattern of AChE-li staining. One notable exception was the presence of AChE-li cell bodies. In contrast, ChAT immunoreactivity was associated only with fibers and terminals. NGFR-li staining corresponded closely to the ChAT-li fiber pattern. ChAT-li fibers in the CA fields diffusely filled the stratum pyramidale and extended into the stratum oriens and radiatum as well. The highest density was consistently observed in CA4 and CA3 subfields. Staining decreased from CA4 to CA1 and was substantially less dense in the subicular complex. In the entorhinal cortex, the ChAT- and NGFR-li fiber innervation displayed a laminar pattern, most intense over the nests of cells in layer II. There was a trend towards an age-related reduction in the density of ChAT- and AChE-li fibers and terminals. Nonetheless, we also found a surprisingly conserved NGFR-li innervation and the presence of occasional NGFR-li pyramidal cells, providing evidence of a plastic response in the brains of the elderly patients.

Expression of choline acetyltransferase and nerve growth factor receptor within hypoglossal motoneurons following nerve injury.

In the present study we employed light microscopic immunocytochemical techniques in order to investigate the temporal response of choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr) within hypoglossal motoneurons following unilateral transection or crushing of the XII nerve or after intraneural injections of ricin into the nerve. In control rats (i.e., sham operated) virtually all the motoneurons of the XII nucleus displayed intense immunolabeling for ChAT and were devoid of NGFr immunoreactivity. As early as 3 days post-operative the intensity and the number of ChAT-labeled neurons were reduced on the axotomized side compared to the non-lesioned side. This decrease was maximal approximately two weeks post-operative when virtually no ChAT-labeled cells were present on the lesioned side. In contrast, no loss of hypoglossal neurons was found using Nissl stains. This absence of ChAT immunolabeling persisted for several days, yet by 30 days many of the motoneurons had begun to re-express the enzyme. In contrast to the decrease in ChAT immunoreactivity, transection of the XII nerve also resulted in the expression of NGFr immunoreactivity within the lesioned motoneurons. This response was detected as early as one day post-operatively and continued throughout all time points thus far examined including times after many of the motoneurons had begun to re-express ChAT. Crushing of the XII nerve effected the expression of ChAT and NGFr in a manner comparable to, yet less intense than, that observed following transection. Ricin injected directly into the XII nerve resulted in the loss of hypoglossal motoneurons as demonstrated both in immunohistochemical and Nissl-stained tissue preparations. The cell loss was readily apparent 3 days post-operatively, and ChAT immunoreactivity permanently disappeared. NGFr immunolabeling was seen only in scattered surviving neurons but not in ricin poisoned cells. The possible mechanisms underlying the differential expression of ChAT and NGFr are discussed.

Prefrontal cortical projections to the cholinergic neurons in the basal forebrain.

The prefrontal cortex (PFC) projections to the basal forebrain cholinergic cell groups in the medial septum (MS), vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB), and the magnocellular basal nucleus (MBN) in the rat were investigated by anterograde transport of Phaseolus vulgaris leuco-agglutinin (PHA-L) combined with acetylcholinesterase (AChE) histochemistry or choline acetyltransferase (ChAT) immunocytochemistry. The experiments revealed rich PHA-L-labeled projections to discrete parts of the basal forebrain cholinergic system (BFChS) essentially originating from all prefrontal areas investigated. The PFC afferents to the BFChS display a topographic organization, such that medial prefrontal areas project to the MS, VDB, and the medial part of the HDB, whereas the orbital and agranular insular areas predominantly innervate the HDB and MBN, respectively. Since the recurrent BFChS projection to the prefrontal cortex is arranged according to a similar topography, the relationship between the BFChS and the prefrontal cortex is characterized by reciprocal connections. Furthermore, tracer injections in the PFC resulted in anterograde labeling of numerous "en passant" and terminal boutons apposing perikarya and proximal dendrites of neurons in the basal forebrain, which were stained for the cholinergic marker enzymes. These results indicate that prefrontal cortical afferents make direct synaptic contacts upon the cholinergic neurons in the basal forebrain, although further analysis at the electron microscopic level will be needed to provide conclusive evidence.

Response of septal cholinergic neurons to axotomy.

In the present study we employed quantitative morphometric techniques to assay the response of septal cholinergic neurons following unilateral transection of the fimbria/fornix and supracallosal stria. Analysis of 50-micron-thick tissue sections with a Quantimet 920 image analysis system demonstrated a reduction in ChAT immunoreactivity as early as 1 day following denervation. This decrease was associated with a drop in the number of labeled cells ipsilateral to the lesion and a decrease in the area of cholinergic perikarya on the lesioned and nonlesioned side of the septum. The response at 1 day, however, was transient, and at 4 days the number of labeled neurons was not significantly different from controls. By 8 days we observed a dramatic reduction in the number and size of ChAT-positive cells ipsilateral to the lesion and a reduction in the size of cholinergic perikarya on the contralateral (i.e., nonlesioned) side. These values persisted throughout the remainder of the study. To assess more completely the morphologic response of neurons to axotomy than can be determined in 50-micron-thick tissue sections, we embedded the adjacent immunolabeled tissue section in Epon and then serially sectioned it to a thickness of 0.75-1.0 micron. By using this method, we were able to measure the area, length, and width of the cell, the area of the nucleus and nucleolus, and the position of the nucleus (i.e., eccentricity). Measurements were performed on ChAT-labeled and nonlabeled cells. The results of our studies demonstrate that cholinergic and noncholinergic cells responded to axotomy in a characteristic yet different fashion from each other and that this response could be quantitatively assayed. In general, labeled and nonlabeled cells on the lesioned side of the septum shrink in response to denervation. This shrunken state was reflected in measurements of cellular area, length, width, and nuclear area. Moreover, other measurements of cellular morphology (i.e., area of the nucleolus, position of the nucleus) indicate that none of the neuronal populations examined in the present study displayed morphologic evidence of regeneration. Our results indicate a dramatic loss of cholinergic perikarya ipsilateral to the lesion. Moreover, although a few neurons do persist they do so in a shrunken state. These data provide an essential baseline for the second study in this series, which will evaluate the effect of nerve growth factor on the survival of denervated septal neurons.

Heterogeneity and selectivity of the degeneration of cholinergic neurons in the basal forebrain of patients with Alzheimer's disease.

Cholinergic neurons were studied by immunohistochemistry, with an antiserum against choline acetyltransferase (ChAT), in the basal forebrain (Ch1 to Ch4) of four patients with Alzheimer's disease (AD) and four control subjects. ChAT-positive cell bodies were mapped and counted in Ch1 (medial septal nucleus), Ch2 (vertical nucleus of the diagonal band), Ch3 (horizontal nucleus of the diagonal band) and Ch4 (nucleus basalis of Meynert). Compared to controls, the number of cholinergic neurons in AD patients was reduced by 50% on average. The interindividual variations in cholinergic cell loss were high, neuronal loss ranging from moderate (27%) to severe (63%). Despite the small number of brains studied, a significant correlation was found between the cholinergic cell loss and the degree of intellectual impairment. To determine the selectivity of cholinergic neuronal loss in the basal forebrain of AD patients, NPY-immunoreactive neurons were also investigated. The number of NPY-positive cell bodies was the same in controls and AD patients. The results (1) confirm cholinergic neuron degeneration in the basal forebrain in AD and the relative sparing of these neurons in some patients, (2) indicate that degeneration of cholinergic neurons in the basal forebrain contributes to intellectual decline, and (3) show that, in AD, such cholinergic cell loss is selective, since NPY-positive neurons are preserved in the basal forebrain.

Preservation of nucleus basalis neurons containing choline acetyltransferase and the vesicular acetylcholine transporter in the elderly with mild cognitive impairment and early Alzheimer's disease.

Immunocytochemistry for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) was used to examine the expression of these linked cholinergic markers in human basal forebrain, including cases with early stages of Alzheimer's disease (AD). Previous neurochemical studies have measured decreased ChAT activity in terminal fields, but little change or even increased levels of VAChT. To determine total cholinergic neuron numbers in the nucleus basalis of Meynert (nbM), stereologic methods were applied to tissue derived from three groups of individuals with varying levels of cognition: no cognitive impairment (NCI), mild cognitive impairment (MCI), and early-stage Alzheimer's disease (AD). Both markers were expressed robustly in nucleus basalis neurons and across all three groups. On average, there was no significant difference between the number of ChAT- (210,000) and VAChT- (174, 000) immunopositive neurons in the nbM per hemisphere in NCI cases for which the biological variation was calculated to be 17%. There was approximately a 15% nonsignificant reduction in the number of cholinergic neurons in the nbM in the AD cases with no decline in MCI cases. The number of ChAT- and VAChT-immunopositive neurons was shown to correlate significantly with the severity of dementia determined by scores on the Mini-Mental State Examination, but showed no relationship to apolipoprotein E allele status, age, gender, education, or postmortem interval when all clinical groups were combined or evaluated separately. These data suggest that cholinergic neurons, and the coexpression of ChAT and VAChT, are relatively preserved in early stages of AD.