J.R. McDermott

Brain aluminum in aging and Alzheimer disease.

Aluminum was assayed by atomic absorption spectroscopy in 274 brain samples, and assayed in neurons isolated in bulk from the frontal cortex of patients with Alzheimer dementia and from age-matched patients with no neurologic disease. Brain aluminum concentration increased with age, from late middle age to old age. There were no statistically significant differences in brain aluminum concentration between the 10 patients with Alzheimer disease (mean, 2.7 μg per gram dry weight of tissue; mean age, 81 years), and the 9 nonneurological controls (mean, 2.5 μg per gram; mean age, 73 years). In both groups, the hippocampus had the highest concentration of aluminum (5.6 μg per gram), and the corpus callosum the lowest (1.5 μg per gram).

Mechanism of neurotensin degradation by rat brain peptidases.

Neurotensin is degraded by peptidases present in soluble and particulate (25000 x g) fractions of rat hypothalamus, thalamus, cortex and pituitary, the soluble fraction of the hypothalamus having the highest activity. High performance liquid chromatography and amino acid analysis were used to identify the degradation pathway. The main product in both fractions was [1-8]neurotensin and the corresponding C-terminal fragment [9-13]neurotensin was identified. [1-10]Neurotensin was also identified, proportionately more of this peptide being produced by the particulate rather than the soluble fraction. In the presence of dithiothreitol, [1-7]neurotensin and [1-10]neurotensin were major products particularly in the soluble fraction. These results suggest that the main sites of cleavage of neurotensin by rat brain peptidases are the Arg8-Arg9, Pro10-Tyr11 and Pro7-Arg8 bonds.

Degradation of Alzheimer's beta-amyloid protein by human cathepsin D.

The aim of the study was to identify and characterize human brain peptidases capable of degrading Alzheimers beta-amyloid protein. Synthetic beta-amyloid protein (1-40) was rapidly degraded by a human brain soluble fraction, optimum activity occurring at around pH4. Pepstatin totally inhibited the activity showing that an aspartyl protease was responsible. HPLC separation and identification of the degradation products showed that the L34-M35 bond was the primary site of cleavage followed by hydrolysis of the F19-F20 and F20-A21 bonds. The major lysosomal aspartyl protease, cathepsin D, hydrolysed beta-amyloid protein with the same pH profile, inhibitor sensitivity and bond specificity as the activity present in human brain soluble fraction. We suggest that cathepsin D may play an important role in regulating brain concentrations of beta-amyloid protein (1-40).

Post mortem levels of thyrotropin-releasing hormone and neurotensin in the amygdala in Alzheimer's disease, schizophrenia and depression

The levels of neurotensin and thyrotropin-releasing hormone (TRH) in normal post mortem human amygdala have been compared with those in cases of schizophrenia, Alzheimers disease and depression. Amongst various factors which can influence post mortem human brain biochemistry (including age, sex, post mortem delay, time of death, disease status and severity), sex difference appeared to be responsible for the most extensive variation. The levels of both peptides were nearly doubled in males compared with females and this increase was significant in the case of neurotensin. There was also a positive correlation between neurotensin and TRH levels. Although levels of neurotensin and TRH tended to be lower in the disease groups these trends did not reach significance.

Somatostatin immunoreactivity in cortical and some subcortical regions in Alzheimer's disease.

Reverse phase HPLC analysis of somatostatin immunoreactivity in the cerebral cortex in elderly normal individuals revealed that the majority of the immunoreactivity co-eluted with synthetic somatostatin-14. While an immunoreactive peak corresponding to somatostatin-28 was not detected there was a peak of immunoreactivity which eluted after somatostatin-14. In cases of senile dementia of Alzheimer type (SDAT), where abundant neurofibrillary tangles and senile plaques (density greater than 30 per 1.3-mm2 field) were present in the cerebral cortex, somatostatin immunoreactivity was found to be significantly decreased in either the frontal or temporal cortex. Chromatographic analysis, however, revealed that both the major immunoreactive peaks detected in the normal group were reduced in SDAT in the temporal and frontal cortex. Using a punch microdissection technique somatostatin immunoreactivity has been assessed in the nucleus of Meynert and amygdala of SDAT and elderly normal cases. While there was no change in somatostatin immunoreactivity in the nucleus of Meynert in the SDAT group, tissue punches taken from the amygdala revealed a selective decrease in somatostatin immunoreactivity in the basal nucleus, in the SDAT cases.

Purification and characterization of a neuropeptide-degrading aminopeptidase from human brain

Abstract: The major aminopeptidase from human post‐mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion‐exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2‐mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal‐ion chelating agents, sulphhydryl blocking agents, bestatin, and puromycin. A series of amino acyl‐7‐amido‐4‐methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 μM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5‐enkephalin (Km 180 μM), cholecystokinin octapeptide, and Arg8‐vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5‐enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis.

Characterization of corticotropin-like immunoreactive peptides in rat brain using high performance liquid chromatography

The molecular nature of corticotropin (ACTH)-related peptides in rat brain has been studied using high performance liquid chromatography (HPLC) and radioimmunoassay. The major ACTH-immunoreactive species in rat hypothalamic extracts coelutes with corticotropin-like intermediate lobe peptide (CLIP); ACTH18-39) on two HPLC solvent gradients, and has 3-4 times more C-terminal than N-terminal immunoreactivity. N-terminal ACTH-immunoreactivity is composed of a number of peaks on HPLC with less than 10% eluting at the position of ACTH. Hypothalamic C-terminal ACTH immunoreactivity is also heterogeneous and resembles in some respect that seen in the rat neuro-intermediate lobe. Around 90% of the alpha-MSH immunoreactivity in the hypothalamus elutes as a single peak in the position of des[N-acetyl] alpha -MSH (ACTH1-13-NH2).

Purification and Characterization of Two Soluble C1−-Activated Arginyl Aminopeptidases from Human Brain and Their Endopeptidase Action on Neuropeptides

Abstract: Two closely related Cl− ‐activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion‐exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of 1 and II on aminoacyl‐7‐amido‐4‐methyl‐coumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peg tides including neurotensin, bradykinin, angiotensin‐I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu‐enkephalin, proctolin, thyroliberin, adrenocorticotropin18–39 (ACTH18–39), ACTH11–24, and dynorphin (1–13)] were not appreciably hydrolysed. The amino‐ and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The ami‐nopeptidase activity was stimulated 20‐fold by Cl− ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 μM (I) and 37 μM (II) and for Arg‐AMC hydrolysis they were 167 μM (I) and 125 μM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphame‐nine or bestatin (IC50= 9 nM and 0.1 μM, respectively, with Arg‐AMC substrate).

Biotransformation of neuropeptides

The ability of neuropeptides to act as precursors for smaller, biologically active fragments is discussed in terms of their biotransformation. This process may involve cleavage of the parent peptide by peptidase enzymes to produce shorter polypeptides with defined biological activity, though other enzymic processes such as sulphation and acetylation may be implicated. Detection of the specific fragments in vivo, their release, receptor-binding and biological actions may confirm biotransformation of the parent peptide. Control of biotransformation will depend upon the localization, both regional and subcellular, and the specificity of the enzymes involved. This process may give an additional degree of flexibility to the biological effects of neuropeptides.

Studies on the characterisation of α-MSH-like immunoreactivity in rat hypothalamus

Abstract Two distinct cell groups contain α-melanocyte-stimulating hormone-like immunoreactivity in rat hypothalamus. Only one group, located in the arcuate nucleus, contains other opiocortin peptide immunoreactivity. Combined immunocytochemistry, radioimmunoassay and high performance liquid chromatography, using two different antisera, were used in an attempt to characterise the immunoreactive material present in each cell group. The results thus obtained from normal rats, using an antiserum against α-melanocyte-stimulating hormone and one against COOH-terminal adrenocorticotropin, were compared with those obtained from rats treated neonatally with monosodium glutamate, which destroys the arcuate nucleus. In animals treated with monosodium glutamate, cells of the arcuate nucleus, staining with both antisera, were reduced in number. Cells containing only α-melanocyte-stimulating hormone-like immunoreactivity in the lateral hypothalamus were unaffected. Peptide levels detected by radioimmunoassay with both antisera were reduced in parallel. Chromatographed extracts showed parallel reductions in α-melanocyte-stimulating hormone-like and COOH-terminal adrenocorticotropin-like immunoreactivities. These results suggest that if the immunostained cells of the lateral hypothalamus contain conventional α-melanocyte-stimulating hormone, it constitutes only a very small proportion of the total hypothalamic concentration. However, the possibilities that the antiserum is crossreacting with a different molecular species, or with a similar compound synthesised by a different pathway cannot be excluded.