Cellular and ultra structural evidence for cytoskeletal localization of prolyl endopeptidase-like protein in neurons.

The biochemical properties and subcellular localization of prolyl endopeptidase (PREP) in brain are well characterized and its implications in the realization of cognitive processes and in the pathogenesis of neurodegenerative disorders are a matter of intensive investigation. In contrast, very little is known about its homolog, the PREP-like protein (PREPL). In order to obtain initial hints about the involvement of PREPL in physiological processes, a differential proteomic screen was performed with human skin fibroblasts from controls and patients with PREPL deficiency (hypotonia-cystinuria syndrome). The majority of affected proteins represented cytoskeletal proteins, including caldesmon, tropomyosin α3 chain, lamin A, ß-actin, γ-actin, vimentin and zyxin. Therefore, the analysis of PREPL subcellular localization by confocal laser scanning and electron microscopy in mouse neurons was focused on the cytoskeleton. The co-localization of PREPL with cytoskeletal marker proteins such as ß-actin and microtubulin-associated protein-2 was observed, in addition to the presence of PREPL within Golgi apparatus and growth cones. In the mouse brain, PREPL is neuronally expressed and highly abundant in neocortex, substantia nigra and locus coeruleus. This mirrors to some extent the distribution pattern of PREP and points toward redundant functions of both proteins. In the human neocortex, PREPL immunostaining was found in the cytoplasm and in neuropil, in particular of layer V pyramidal neurons. This staining was reduced in the neocortex of Alzheimer's disease (AD) patients. Moreover, in AD brains, PREPL immunoreactivity was observed in the nucleus and in varicose neuritic processes. Our data indicate physiological functions of PREPL associated with the cytoskeleton, which may be affected under conditions of cytoskeletal degeneration.

Role of prolyl endopeptidase in intracellular transport and protein secretion.

Prolyl endopeptidase (E.C. 3.4.21.26, PREP) also known as prolyl oligopeptidase is an enzyme which cleaves several peptides at the carboxyl side of proline residues. Since brain contains relatively large amounts of this enzyme and because of its substrate specificity it has been suggested to play a role in the metabolism of neuropeptides, acting both on their maturation and their degradation. The final step of neuropeptide processing occurs in the synaptic vesicles and degradation of most of these peptides takes place in the synaptic cleft. Thus, a localization of PREP in these cellular compartments appears to be feasible. Here we summarize recent data and provide novel evidence for the subcellular localization of PREP. Most importantly, immunocytochemical double labelling, confocal laser scanning and electron microscopic procedures as well as functional assays strongly suggest a role for PREP in intracellular transport mechanisms and in protein secretion.

Cerebrospinal fluid cholinesterases--markers for loss of cholinergic basal forebrain neurons?

The present study was conducted to test the hypothesis that cholinergic basal forebrain neurons are a major source of cerebrospinal fluid (CSF) cholinesterases. To address this question enzyme activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in both CSF and parietal cortex were assayed following selective lesion of basal forebrain cholinergic neurons by a single intracerebroventricular application of the cholinergic immunotoxin 192IgG-saporin. Cholinergic immunolesions led to a dramatic decrease in total AChE activity in parietal cortex, which was due to the specific loss of the G4 molecular form while the activity of the G1 form was increased as compared to nonlesioned animals. In contrast, the total enzyme activity of BChE and its molecular forms were not affected by cholinergic lesion in both parietal cortex and CSF. The data suggest, that cholinergic basal forebrain neurons are seemingly not a major source of cholinesterases in the CSF, and do not provide any evidence for using CSF cholinesterases as a diagnostic marker of basal forebrain cholinergic cell loss in humans.

Cellular distribution of 6-phosphofructo-1-kinase isoenzymes in rat brain.

In the brain, all three isoenzyme types [muscle (M), liver (L), and brain (C)] of 6-phosphofructo-1-kinase (PFK; EC 2.7.1.11) occur, forming a complex mixture of homo- and heterotetramers. The PFK isoenzyme pattern of the different brain cell types is yet unknown. In the present study, we investigated the distribution of the PFK isoenzyme subunits in primary and secondary cell cultures and in bulk-isolated cells of rat brain by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. All three PFK isoenzymes are present in all cell types but in different proportions. The cellular distribution of the PFK isoenzymes in situ was studied immunohistochemically with different polyclonal antisera against purified rat PFKs. The monospecific antibody against M-type PFK stained preferentially the perinuclear areas of neurons and glial cells. The antibodies that in immunoblots detected mainly the L-type PFK showed a characteristic staining in only the cytoplasma and the processes of cells, whereas the C-type antibodies almost homogeneously stained whole cell bodies as well as large dendrites. Because the PFK isoenzymes differ with respect to their allosteric properties, their differential distribution in different brain cells might be of importance for the regulation of brain glycolysis in the different cellular compartments of the brain.

Chymotrypsin-catalyzed peptide synthesis in an acetonitrile-water-system: studies on the efficiency of nucleophiles.

Peptide synthesis with chymotrypsin in organic solvents was investigated and the apparent partition constants have been measured. We find that the Papp values of the most amino acids and peptide derivatives are drastically changed and the stereo- and regiospecificity in acetonitrile/water mixture is reduced.