Gordon Bruce

Immunoaffinity Purification of Human Choline Acetyltransferase: Comparison of the Brain and Placental Enzymes

Abstract: A rapid and efficient immunoaffinity purification procedure has been developed for human placental choline acetyltransferase (ChAT). Using this procedure, human placental ChAT was purified to homogeneity with high recovery of enzyme activity (50–60%). Purified ChAT was used to raise a monospecific anti‐human ChAT polyclonal antibody in rabbits. A comparison of the physical properties of ChAT was made between the enzymes purified from human brain and human placenta. Only one form of the enzyme exists in either tissue, having identical molecular weights of 68,000 and a single apparent pI of 8.1. A more detailed comparison of the two enzymes using peptide mapping and epitope mapping indicates identity between the brain and placental enzymes.

Distribution of choline acetyltransferase-containing neurons of the hypothalamus

A system of small to medium size choline acetyltransferase (ChAT)-containing neurons has been identified in rat, monkey and human hypothalamus. A highly sensitive polyclonal anti-human placental ChAT rabbit serum, combined with a nickel ammonium sulfate second antibody intensification method, was used to identify these relatively weakly staining ChAT-positive neurons. The most prominent hypothalamic group consisted of small neurons in the infundibular (arcuate) nucleus. Fibers extended towards the infundibulum. Other ChAT-positive cells were not identified with specific hypothalamic nuclei but were scattered loosely in the surrounding matrix. They fell into two broad complexes: a medially distributed one close to the third ventricle and running rostrocaudal to caudoventral; and a lateral one distributed principally in the region of the medial forebrain bundle. The most laterally placed hypothalamic ChAT-positive neurons slightly overlapped with the large, intensely staining cells of the medial basal forebrain cholinergic complex. The identification of these cells helps to account for previous biochemical and pharmacological studies which have strongly indicated the presence of intrinsic cholinergic neurons in the hypothalamus.

Immunohistochemical staining of cholinergic neurons in the human brain using a polyclonal antibody to human choline acetyltransferase

Antibodies against human placental choline acetyltransferase (ChAT) were used to immunohistochemically stain cholinergic neurons in the neostriatum and nucleus basalis of Meynert in human brain. Cells in both regions were intensely stained as were nerve fibers. Comparable cells were stained in these same brain regions in the rat. This anti-human ChAT antibody will enable the further detailed characterization of cholinergic neurons in the human brain in both health and disease.

Development of cholinergic neurons in the septal/diagonal band complex of the rat

In the present study we employed immunohistochemical techniques using a polyclonal antibody against choline acetyltransferase (ChAT) to determine the distribution and cytological features of cholinergic neurons in the developing septal/diagonal band complex of the rat. ChAT-positive perikarya were first clearly detected in this region on embryonic day 17, although the neurons were faintly labeled and lacked the cytological details found in the adult. After birth we observed a dramatic increase in the intensity of the immunolabeling which continued until postnatal day 23. Thereafter, the ChAT-positive neurons assumed their adult-like characteristics.

Cholinergic innervation displays strikingly different laminar preferences in several cortical areas

A new rabbit polyclonal antiserum against choline acetyltransferase (ChAT) reveals that cholinergic innervation of the cortex varies strikingly among different cytoarchitecturally defined areas in the rat neocortex. These findings suggest that cholinergic transmission may be integrated differently into the local circuitries of various regions of the cerebral cortex. In addition, the pattern of staining observed with acetylcholinesterase histochemistry, which has been used for many years to demonstrate putative cholinergic fibers, only partially matches the staining pattern obtained with the more specific cholinergic marker, ChAT.

Vestibular and cochlear efferent neurons in the monkey identified by immunocytochemical methods.

Attempts were made to identify vestibular (VEN) and cochlear (CEN) efferent neurons in the squirrel monkey using retrograde transport of horseradish peroxidase (HRP) and immunocytochemical methods. HRP implants in the ampulla of the lateral semicircular duct retrogradely labeled cells of VEN bilaterally and some cells of CEN. VEN located lateral to the rostral part of the abducens nucleus formed a compact collection of cells, all of which were immunoreactive only to antisera for choline acetyltransferase (ChAT). CEN, identified by immunoreactivity to ChAT were located at the hilus of the lateral superior olive (LSO), along the lateral border of the LSO and sparsely near lateral parts of the ventral trapezoid nucleus (VTN). A small number of cells and fibers near the border of the VTN and lateral to the LSO were immunoreactive for leucine enkephalin (L-ENK). Fibers immunoreactive for L-ENK also were identified in the hilus of the LSO. No cells of the superior olivary complex were immunoreactive for antisera to ChAT, L-ENK, substance P, gamma-aminobutyric acid or glutamic acid decarboxylase. Cells of VEN and CEN can be identified by their immunoreactivity to ChAT, and some cells and fibers of CEN also contain L-ENK.

Relation of pontine choline acetyltransferase immunoreactive neurons with cells which increase discharge during REM sleep

The purpose of this study was to determine whether neurons in the medial pontine reticular formation with high discharge rates during REM sleep could be localized in regions of the brainstem having neurons displaying choline acetyltransferase immunoreactivity. Six cats were implanted with sleep recording electrodes and microwires to record extracellular potentials of neurons in the pontine reticular formation. Single-units with a S:N ratio greater than 2:1 were recorded for at least two REM sleep cycles. A total of 49 units was recorded from the pontine reticular formation at medial-lateral planes ranging from 0.8 to 3.7 mm. The greatest proportion of the units (28.6%) showed highest discharge during active waking and phasic REM sleep compared to quiet waking, non-REM sleep, transition into REM sleep or quiet REM sleep periods. A percentage (20.4%) of the cells had high discharge associated with phasic REM sleep periods while 8.2% of the cells showed a progressive increase in discharge from waking to REM sleep. Subsequent examination of the distribution of choline acetyltransferase immunoreactive cells in the PRF revealed that cells showing high discharge during REM sleep were not localized near presumed cholinergic neurons. Indeed, we did not find any ChAT immunoreactive somata in the medial PRF, an area which has traditionally been implicated in the generation of REM sleep. These results suggest that while increased discharge of PRF cells may be instrumental to REM sleep generation, these cells are not cholinergic.

Choline acetyltransferase immunoreactivity in neuritic plaques of Alzheimer brain.

We have observed dystrophic choline acetyltransferase (ChAT)-positive processes surrounding the amyloid core of neuritic plaques in human neocortex, amygdala and hippocampus, using a polyclonal anti-human ChAT antiserum. These data, and those from studies of the aged monkey by other investigators, provide a morphologic counterpart for the biochemical abnormality of the cholinergic system in Alzheimers disease and senile dementia of the Alzheimer type.

The phosphorylation of choline acetyltransferase

Human placental Choline Acetyltransferase (ChAT) has been shown to be phosphorylated in vitro by kinases present in rat brain. Phosphorylation occurs at a single site with the exclusive phosphoamino acid being serine. ChAT phosphorylation was shown to be calcium, and not cyclic nucleotide, dependent and was inhibited by inhibitors of calcium/calmodulin protein kinases including anti-calmodulin anti-sera. ChAT phosphorylation was stimulated by calmodulin (9 fold) and, to a lesser extent, by phosphatidylserine (4 fold). These results indicate the involvement of a calcium/calmodulin and possibly also a calcium/phosopholipid kinase. This finding was confirmed by demonstrating ChAT phosphorylation using both purified multifunctional calcium/calmodulin protein kinase (CaMK) and calcium/phospholipid protein kinase C (PKC) from rat brain. A stoichiometric incorporation of 0.9 mol phosphate/mol ChAT was achieved by CaMK. Phosphorylated ChAT could be isolated from freshly prepared rat brain synaptosomes. The results obtained with this model system support the hypothesis that in vivo a fraction of ChAT exists phosphorylated.

Production and characterization of biologically active recombinant human nerve growth factor.

Nerve growth factor (NGF) is required for the differentiation and maintenance of sympathetic and sensory neurons. In animal models, NGF prevents the death of septal and basal forebrain cholinergic neurons deprived of endogenous NGF, suggesting that NGF may be of benefit in neurodegenerative diseases of humans. However, little is known about NGF in human brain, partly because a sensitive assay for hNGF has been lacking. As a first step toward developing the tools for the study of NGF in humans, recombinant human NGF (rhNGF) was produced by expressing exon 4 of the human NGF gene in COS cells. The expression vector is driven by the adenovirus major late promoter and contains an SV40 origin of replication. NGF was secreted by transiently transfected cells. Conditioned medium was assayed with an enzyme immunoassay (EIA) that utilizes a monoclonal antibody (clone 27/21) against mouse beta-NGF, and contained 15 ng/ml of rhNGF. The rhNGF migrated as a dimer of 26-29 Kd on a gel permeation chromatography column, and stimulated neurite outgrowth and neuropeptide Y mRNA levels in PC12 cells. With optimization, the described expression system is capable of providing sufficient hNGF for research and therapeutic purposes.