Galanin-Like Immunoreactivity Is Increased in the Postmortem Cerebral Cortex from Patients with Alzheimer's Disease

Abstract: Galanin is a peptide that is associated with cholinergic neurons of the basal forebrain and, thus, of interest for the neuropathology of Alzheimer's disease. In the present study, human galanin-like immunoreactivity was measured in postmortem human cerebral cortical tissues by using a homologous radioimmunoassay. In an initial study, six cerebral cortical regions were evaluated from nine elderly controls, 13 neuropathologically verified Alzheimer's disease patients, and 19 elderly schizophrenics. A significant 65% increase in galanin was found in frontal cortex Brodmann area 8 of Alzheimer's disease patients compared with controls. In contrast, cerebral cortical tissues from elderly schizophrenics were not different from those from elderly controls in any region. In a second study, 10 cerebral cortical regions were evaluated from 50 neuropathologically verified Alzheimer's disease patients and nine elderly controls. Concentrations of galanin were increased significantly 26–61% in six of 10 cerebral cortical regions examined (Brodmann areas F8, F44, T20, T21, T36, and P22). Purification of brain extracts by size-exclusion Sephadex G-50 chromatography revealed that human galanin-like immunoreactivity eluted in two peaks of different molecular weights. These studies reveal increased concentrations of galanin in the cerebral cortex of Alzheimer's disease, similar to previous findings in basal forebrain tissue. Because galanin inhibits cholinergic neurotransmission, these findings may have important implications in the understanding of Alzheimer's disease neuropathology and associated cognitive deficits.     

Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system

The distribution of galanin-like immunoreactivity in various regions of the central nervous system was assessed in three mammalian species, pig, rat, and human, by radioimmunoassay. Galanin concentrations were highest in the hypothalamus and pituitary region. In spinal cord, there was a rostrocaudal/dorsoventral gradient with highest levels observed in the sacral dorsal horn. Serial dilutions of porcine tissue extracts diluted parallel to the porcine standard curve, while the rat and human tissue extracts did not. In all tissues examined by high pressure liquid chromatography, the principal peak of immunoreactivity coeluted with the authentic porcine galanin standard and was decreased by trypsin cleavage. These results suggest a role for galanin in the central nervous system and support species differences in the structure of galanin.     

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.     

Regional distribution of vasoactive intestinal peptide in brains from normal and parkinsonian subjects

The distribution of vasoactive intestinal peptide (VIP) in the post-mortem human brain was determined by radioimmunoassay using a highly specific antiserum. The detection limit of the assay was 4 fmol/tube. The highest concentrations of VIP were found in the cerebral cortex, amygdala, hypothalamus and hippocampus. The lowest levels of peptide were detected in basal ganglia including caudate nucleus, external pallidum, putamen and substantia nigra. All dilution curves of acetic acid extracts from different brain areas were strictly parallel to the standard curve. Sephadex G-50 gel filtration of frontal cortex extract showed that VIP-like immunoreactivity (VIP-LI) eluted as a major peak comigrating with synthetic hVIP. Detailed mapping of VIP in the human cerebral cortex showed the existence of a rostro-caudal gradient of VIP-LI concentrations: the frontal cortex exhibited the highest VIP levels, the parietal and temporal cortex contained medium values and the occipital cortex contained the lowest VIP levels. The concentrations of VIP-LI were compared in various regions of the human brain from normal and parkinsonian subjects. No significant changes in VIP-LI levels occurred in the brains of patients dying with Parkinson's disease. No difference in VIP levels could be found either when the parkinsonian group was subdivided into nondemented and demented patients. These data indicate that VIP-containing neurons are not affected in parkinsonian patients. Our results also suggest that VIP neuronal systems are not involved in the course of dementing process in Parkinson's disease.     

gamma 2-[corrected]-MSH-like immunoreactivity in porcine pituitary and adrenal medulla. An immunochemical and immunocytochemical study

A sensitive and specific radioimmunoassay for gamma 2-melanotropin (gamma 2-MSH) has been developed that does not recognize alpha-, beta-, gamma 1- or gamma 3-MSH. gamma 2-MSH-like immunoreactivity could be demonstrated in the porcine pituitary and adrenal gland. The highest concentrations were detected in the neurointermediate lobe regardless of extraction procedure. The anterior lobe harboured lower concentrations and in adrenal gland extracts only small amounts were measured. Gel chromatography and high performance liquid chromatography of extracts of both pituitary and adrenal gland revealed several peaks of immunoreactive material, one of which eluted close to the position of synthetic gamma 2-MSH. By immunocytochemistry gamma 2-MSH immunoreactivity was localized to the adrenocorticotropin/alpha-MSH cells in the pituitary and to a subpopulation of the noradrenaline-storing cells in the adrenal medulla. Together, the immunocytochemical and immunochemical findings indicate the existence of gamma 2-MSH-like material in the porcine pituitary and adrenal medulla.     

Reduction in brain immunoreactive corticotropin-releasing factor (CRF) in spontaneously hypertensive rats

The brain CRF concentration of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) was examined by rat CRF radioimmunoassay. Anti-CRF serum was developed by immunizing rabbits with synthetic rat CRF. Synthetic rat CRF was also used as tracer and standard. The displacement of 125I-rat CRF by serially diluted extracts of male Wistar rats hypothalamus, thalamus, midbrain, pons, medulla oblongata, cerebral cortex, cerebellum and neurointermediate lobe was parallel to the displacement of synthetic rat CRF. In both WKY and SHR the highest levels of CRF immunoreactivity were shown by the hypothalamus and neuro-intermediate lobe, and considerable CRF immunoreactivity was also detected in other brain regions. The CRF immunoreactivity in the hypothalamus, neurointermediate lobe, midbrain, medulla oblongata and cerebral cortex was significantly reduced in SHR and it may suggest that CRF abnormality may be implicated in the reported abnormalities in the pituitary-adrenal axis, autonomic response and behavior of SHR.     

Purification and sequence determination of canine relaxin

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C₁₈ column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.     

Galanin-Like Immunoreactivity Is Unchanged in Alzheimer''s Disease and Parkinson''s Disease Dementia Cerebral Cortex

Galanin is a recently isolated neuropeptide that is of particular interest in dementing disorders because of its known colocalization with choline acetyltransferase in magnocellular neurons of the basal nucleus of Meynert. These neurons degenerate in Alzheimer's disease, and there is a corresponding deficiency of cortical choline acetyltransferase activity. In the present study, galanin-like immunoreactivity was measured in the postmortem cerebral cortex and hippocampus of 10 controls and 14 patients who had had Alzheimer's disease. Significant reductions of choline acetyltransferase activity (50-60%) were found in all regions examined; however, there was no significant effect on concentrations of galanin-like immunoreactivity. Similar measurements were made in postmortem tissues of 12 control and 13 demented Parkinsonian patients who had had Alzheimer-type cortical pathology. Choline acetyltransferase activity was again significantly decreased in all regions examined but there were no significant reductions in galanin-like immunoreactivity. Experimental lesions of the fornix in rats produced parallel significantly correlated reductions of both choline acetyltransferase activity and galanin-like immunoreactivity in the hippocampus. Galanin-like immunoreactivity in the human hypothalamus consisted of two molecular-weight species on gel-permeation chromatography, and two forms were resolved by reverse-phase HPLC. The paradoxical preservation of galanin-like immunoreactivity, despite depletion of the activity of choline acetyltransferase, with which it is colocalized, is as yet unexplained. Recent studies have shown that galanin inhibits both acetylcholine release in the hippocampus and memory acquisition; therefore, preserved galanin may exacerbate the cholinergic and cognitive deficits that accompany dementia.     

Localization and identification of Neuropeptide Y (NPY)-like immunoreactivity in the frog brain

The distribution of neuropeptide Y (NPY) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. NPY-like containing perikarya were localized in the infundibulum, mainly in the ventral and dorsal nuclei of the infundibulum, in the preoptic nucleus, in the posterocentral nucleus of the thalamus, in the anteroventral nucleus of the mesencephalic tegmentum, in the part posterior to the torus semicircularis, and in the mesencephalic cerebellar nucleus. Numerous perikarya were also distributed in all cerebral cortex. Important tracts of immunoreactive fibers were found in the infundibulum, in the preoptic area, in the lateral amygdala, in the habenular region, and in the tectum. The cerebral cortex was also densely innervated by NPY-like immunoreactive fibers. A rich network of fibers was observed in the median eminence coursing towards the pituitary stalk. Scattered fibers were found in all other parts of the brain except in the cerebellum, the nucleus isthmi and the torus semicircularis, where no immunoreactivity could be detected. NPY-immunoreactive fibers were observed at all levels of the spinal cord, with particularly distinct plexus around the ependymal canal and in the distal region of the dorsal horn. At the electron microscope level, NPY containing perikarya and fibers were visualized in the ventral nuclei of the infundibulum, using the peroxidase-antiperoxidase and the immunogold techniques. NPY-like material was stored in dense core vesicles of 100 nm in diameter. A sensitive and specific radioimmunoassay was developed. The detection limit of the assay was 20 fmole/tube. The standard curves of synthetic NPY and the dilution curves for acetic acid extracts of cerebral cortex, infundibulum, preoptic region, and mesencephalon plus thalamus were strictly parallel. The NPY concentrations measured in these regions were (pmole/mg proteins) 163±8, 233±16, 151±12 and 60±13, respectively. NPY was not detectable in cerebellar extracts. After Sephadex G-50 gel filtration of acetic acid extracts from whole frog brain, NPY-like immunoreactivity eluted in a single peak. Reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay were used to characterize NPY-like peptides in the frog brain. HPLC analysis revealed that infundibulum, preoptic area and telencephalon extracts contained a major peptide bearing NPY-like immunoreactivity. The retention times of frog NPY and synthetic porcine NPY were markedly different. HPLC analysis revealed also the existence, in brain extracts, of several other minor components cross-reacting with NPY antibodies. These results provide the first evidence for the presence of NPY in the brain of a non-mammalian chordate and indicate that the structure of NPY is preserved among the vertebrate phylum. The abundance of NPY producing neurons in the hypothalamus and telencephalon suggests that this peptide may play both neuroendocrine and neurotransmitter functions in amphibians.     

Characterization and distribution of bombesin-like peptides in the rat brain and gastrointestinal tract

Extracts of rat brain and gastrointestinal tract, analyzed by reverse-phase high-performance liquid chromatography and radioimmunoassay, contained two bombesin-like immunoreactivity peaks with similar retention times as porcine gastrin-releasing peptide (GRP) and its COOH-terminal decapeptide, neuromedin C or GRP(18-27). However, the GRP-like peptide peak did not elute with exactly the same retention time as porcine GRP. The highest concentration of bombesin-like immunoreactivity was found in extracts of antrum, whereas the lowest was found in whole brain. Neuromedin C was present at lower concentrations than the GRP in antrum, duodenum, and ileum, while similar amounts of each were found in brain.     

Taurine Levels in Discrete Brain Nuclei of Rats

Concentrations of taurine have been measured in 44 microdissected rat brain nuclei or areas. Taurine is ubiquitously present and distributed unevenly in the rat brain: the ratio of the highest (pyriform cortex) to lowest (midbrain reticular formation) concentrations is 4.7:1. High taurine levels were found in cerebral cortical areas, caudate-putamen, cerebellum, median eminence, and supraoptic nucleus. Acute pain stress reduced taurine levels in the hypothalamus and the lower brainstem nuclei but not in cortical areas. Increased locomotor and behavioral activities following a high dose of amphetamine elevated taurine concentrations significantly in the substantia nigra and locus ceruleus.     

NPY-like peptides occur in the nervous system and midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata

The distribution of the NPY-like substances in the nervous system and the midgut of the migratory locust, Locusta migratoria and in the brain of the grey fleshfly, Sarcophaga bullata was determined by immunocytochemistry using an antiserum directed against synthetic porcine NPY. The peroxidase-antiperoxidase procedure revealed that NPY immunoreactive cell bodies and nerve fibers were observed in the brain, optic lobes, corpora cardiaca, suboesophageal ganglion and ventral nerve cord of the locust and in the brain, optic lobes and suboesophageal ganglion of the fleshfly. In the locust midgut, numerous endocrine cells and nerve fibers penetrating the outer musculature contained NPY-like immunoreactivity. The concentrations of NPY immunoreactive material in acetic acid extracts of locust brain, optic lobes, thoracic ganglia, ovaries and midguts was measured using a specific radioimmunoassay technique. The dilution curves of the crude tissue extracts were parallel to the standard curve. The highest amount of NPY-like immunoreactivity was found in the locust ovary and midgut. Reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay were used to characterize the NPY-like substances in the locust brain and midgut. HPLC-analysis revealed that NPY-immunoreactivity in the locust brain eluted as three separate peaks. The major peak corresponded to a peptide less hydrophobic than synthetic porcine NPY. RP-HPLC analysis of midgut extracts revealed the presence of an additional NPY-immunoreactive peak which had a retention time similar to the porcine NPY standard. The present data show the existence of a widespread network of NPY immunoreactive neurons in the nervous system of the locust and the fleshfly. Characterization of the immunoreactive substances indicates that peptides similar but not identical to porcine NPY are present in the central nervous system and midgut of insects.     

Chromatographic pattern of gut glucagon-like immunoreactivity (GLI) in plasma before and during glucose absorption.

In this work we have studied the chromatographic pattern on Bio-Gel P-30 columns of the glucagon-like immunoreactivity (GLI) present in unextracted plasma from normal dogs in the basal state and after intraduodenal administration of glucose. Basal plasma GLI, measured by R-8 antiserum, was distributed in four distinct fractions, whose approximate molecular weights were: greater than 30000 delta (Fraction I), 10000 delta (Fraction II), 3500 delta (Fraction III) and 2000 delta (Fraction IV). Fraction I accounted for the highest percent of total immunoreactivity. The increase in plasma GLI during glucose absorption was due to a significant increase of Fraction II, which may well correspond to tissue GLI Peak I, while no significant changes were evident in the other three fractions. The fact that tissue Peak I (or plasma Fraction II) ssems to be the factor secreted during glucose absorption puts the material/s of this molecular size in the first place for further investigation.     

Concentrations of vitamin E in various neuroanatomical regions and subcellular fractions,and the uptake of vitamin e by specific areas,of rat brain

Vitamin E concentrations were determined by high-performance liquid chromatography in different anatomical regions of the brain from 3-month-old Fischer 344 rats. Gray matter from cerebellum and cervical spinal cord contained the lowest concentrations, while gray matter from the frontal cortex and thalamus had the highest concentrations of vitamin E. Radioactive α-tocopherol injected intravenously into the rat was readily taken up by brain although the level of uptake was very low compared with the liver. The ratios of brain-to-serum radioactivities ranged from 0.011 to 0.016 depending upon the brain region. Cerebellar gray matter is characterized by a low concentration of unlabeled a-tocopherol and a high level of uptake of radioactive a-tocopherol and thus is particularly active in the metabolism of vitamin E. Concentrations of unlabeled a-tocopherol were highest in microsomal and mitochondrial fractions and were the lowest in cytosol and nuclear fractions.     

Time dependent changes in the concentrations of immunoreactives cholecystokinin octapeptide sulfate in rat brain regions after systemic injection of picrotoxin

Cholecystokinin octapeptide sulfate-like immunoreactivity (CCK-8S-LI) was determined by enzyme linked immunoassay (ELISA) in seven rat brain areas following injections subcutanously of the Cl^- channel blocker, picrotoxin. Time dependent changes in the concentrations of CCK-8S-LI were seen in the hippocampus, amygdaloid complex, septum, and hypothalamus at 15–180 min after the injection. Concentrations of CCK-8S-LI in the frontal cortex, striatum, and thalamus did not show significant changes. CCK-8S-LI in the amygdaloid complex and hypothalamus was the lowest concentrations at 60 min, and the concentrations in the hippocampus and septum were the lowest at 180 min. The data on high-performance liquid chromatography of the extracts from rat amygdaloid complex showed that changes in the concentrations of CCK-8S-LI were mainly due to the concentration of CCK-8S itself. These data indicate that systemic injection of picrotoxin decreases the concentration of CCK-8S in the brain regions, and the decreases in the amygdaloid complex and hypothalamus occur earlier than that in the hippocampus and septum.     

"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.     

The use of perfused rat intestine to characterise the glucagon-like immunoreactivity released into serosal secretions following stimulation by glucose.

Isolated perfused intestine of rat was used to demonstrate the glucose-stimulated release of glucagon-like immunoreactivity (GLI) into serosal secretions. The released GLI was characterised using immunoaffinity chromatography on columns of immobilised antibodies specific for the N (residues 1 to 18) and for the C (residues 19-29) terminal portions of glucagon followed by gel-filtration. The immunoreactivity was present in a variety of molecular species. These include a large GLI which has a molecular weight about 12000 and binds to antibodies specific for the N-terminal portion of glucagon and two polypeptide fractions with molecular weight closer to that of glucagon. While one fraction of the small GLI boun both to antibodies specific for the C-terminal and N-terminal portions of glucagon the other bound only to the former antibodies. The relevance of these findings to the origins of circulating GLI and the possible precursor relationship between large and other forms of GLI is discussed.     

Cloning and Spatio-Temporal Expression of Porcine CDK5 and CDK5R1(p35) Genes

Cyclin-dependent kinase 5 (CDK5) is a serine/threonine kinase homologue attributed to the mitotic cyclin-dependent kinase family. Both the kinase activity and the biological effects of CDK5 in central nervous system are mainly dependent on association with its regulatory subunit 1 known as CDK5R1 (p35). In the present study, the full-length coding regions of CDK5 and CDK5R1 were cloned from pigs. Radiation hybrid mapping localized porcine CDK5 to chromosome 18q12-13, whereas CDK5R1 was electro-localized to chromosome 12q12. Real-time quantitative RT-PCR (qRT-PCR) showed that CDK5 mRNA is ubiquitously present in all porcine tissues examined, with relatively high levels in cerebral cortex, cerebellum, testicle and lung. We also examined the expression profile of porcine CDK5/CDK5R1 in various tissues at different developmental stages. The results indicated that CDK5 mRNA reaches the highest level in cerebral cortex at two months of age and in cerebellum and liver at 4 months of age, respectively, whereas the peak level of CDK5R1 was observed in both cerebral cortex and cerebellum at two months of age, indicating the pivotal role of CDK5/CDK5R1 during the development of porcine brain.     

Half life of gastrointestinal glucagon-like immunoreactive materials.

The composition, half life and hyperglycemic action of the porcine gastrointestinal glucagon-like immunoreactive materials were examined. Glucagon immunoreactivity (GI) measured using specific antiglucagon serum was more abundunt in the extract from the gastric fundus than in the one from the small intestine. When the extract from the gastric fundus was injected in dogs, the half life (T1/2) of total glucagon-like immunoreactivity (total GLI) measured using nonspecific antiglucagon serum was 9.5 +/- 1.1 min (mean +/- SEM), which was longer than that of crystalline pancreatic glucagon, 3.4 +/- 0.2 min, but shorter than that of the extract from the small intestine, 15.9 +/- 1.3 min. On the other hand, T1/2 for GI from the gastric fundus was 5.1 +/- 0.9 min, which was not significantly different from that of crystalline pancreatic glucagon. Blood sugar levels were significantly increased from the basal by 25 +/- 4 mg/100 ml at 10 min and 19 +/- 4 mg/100 ml at 15 min following an injection of the extract from the gastric fundus, but such a change in blood sugar levels was not demonstrated when the extract from the small intestine was injected. These results suggest that GI of the gastric fundus is close to pancreatic glucagon in respect of its metabolism and hyperglycemic activity.     

Highly sensitive assay for phenylethanolamine N-methyl-transferase activity in rat brain by high-performance liquid chromatography with electrochemical detection

This paper describes a new and highly sensitive assay for phenylethanolamine N-methyl-transferase (PNMT) activity with noradrenaline as substrate in various rat brain regions by high-performance liquid chromatography with electrochemical detection. Commercially available noradrenaline contained about 0.27% of contaminating adrenaline, which was removed to reduce the blank value. Enzymatically formed adrenaline was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid, separated by high-performance reversed-phase paired-ion chromatography and measured with electrochemical detection. 3,4-Dihydroxybenzylamine was added to the incubation mixture as an internal standard after the reaction. This assay was very sensitive and 0.5 pmol of adrenaline formed enzymatically could be detected. This assay method was applied to measure PNMT activity in various rat brain regions. The highest activity was observed in the hypothalamus, pons plus medulla oblongata, septum, lower brain stem, and cerebral cortex; the lowest activity was in the striatum, hippocampus, cerebellum, and limbic brain.