Changes in choline acetyltransferase activity and high-affinity choline uptake, but not in acetylcholinesterase activity and muscarinic cholinergic receptors, in rat somatosensory cortex after sciatic nerve injury

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.     

Cholinergic Control of Nerve Growth Factor in Adult Rats: Evidence from Cortical Cholinergic Deafferentation and Chronic Drug Treatment

Abstract: It is well documented that nerve growth factor (NGF) plays an important role in maintaining functions of cholinergic basal forebrain neurons. In the present study, we tested the hypothesis that cholinergic activity controls NGF levels in cholinoceptive neurons of the cerebral cortex and hippocampus. To address that question, we used both cholinergic deafferentation of cerebral cortex and hippocampus by cholinergic immunolesion with 192IgG-saporin and chronic pharmacological treatment of sham-treated and immunolesioned rats with the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine. We observed an increase in NGF protein levels in the cortex and hippocampus after cholinergic immunolesions and also after muscarinic receptor blockade by chronic intracerebroventricular scopolamine infusion in sham-treated rats after 2 weeks. There was no further increase in the accumulation of NGF after scopolamine treatment of immunolesioned rats. Chronic infusion of pilocarpine had no effect on cortical and hippocampal NGF protein levels in sham-treated rats. In rats with cholinergic immunolesions, however, pilocarpine did prevent the lesion-induced accumulation of NGF. There was no effect of cholinergic lesion and drug treatment on cortical or hippocampal NGF mRNA levels, consistent with the importance of NGF retrograde transport as opposed to its de novo synthesis. This study provides strong evidence for the hypothesis that there is cholinergic control of cortical and hippocampal NGF protein but not mRNA levels in adult rats.     

Effect of visual deprivation on neurotransmitter receptor development in the rat visual system

The effect of monocular deprivation (covering one eye on the 11th posnatal day) and rearing in darkness (the animals were born, developed, and killed with total exclusion of light) on development of -adrenergic, serotoninergic, muscarinic cholinergic, glutamatergic, and benzodiazepine receptors in primary visual structures (visual cortex, lateral geniculate body, superior colliculus, and retina) was investigated in rats. For quantitative analysis of neurotransmitter receptors, the technique of ligand binding was used. It was shown that monocular deprivation affects the development of -adrenergic, serotoninergic, and glutaminergic receptors mainly in the lateral geniculate body and retina, whereas rearing in darkness affects mainly the development of serotoninergic receptors in the superior colliculus. The development of receptors of the cholinergic neurotransmitter system is disturbed only temporarily by visual deprivation. Changes found in receptor development after visual deprivation indicate altered activity of different neurotransmitter systems in corresponding visual structures.P. Flechsig Brain Research Institute, Leipzig, East Germany. Karl Marx University, Leipzig, East Germany. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 691–701, September–October, 1984.     

Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes.

Peroxisomes, glyoxysomes, glycosomes, and hydrogenosomes have each been classified as microbodies, i.e., subcellular organelles with an electron-dense matrix that is bound by a single membrane. We investigated whether these organelles might share a common evolutionary origin by asking if targeting signals used for translocation of proteins into these microbodies are related. A peroxisomal targeting signal (PTS) consisting of the COOH-terminal tripeptide serine-lysine-leucine-COOH has been identified in a number of peroxisomal proteins (Gould, S.J., G.-A. Keller, N. Hosken, J. Wilkinson, and S. Subramani. 1989. J. Cell Biol. 108:1657-1664). Antibodies raised to a peptide ending in this sequence (SKL-COOH) recognize a number of peroxisomal proteins. Immunocryoelectron microscopy experiments using this anti-SKL antibody revealed the presence of proteins containing the PTS within glyoxysomes of cells from Pichia pastoris, germinating castor bean seeds, and Neurospora crassa, as well as within the glycosomes of Trypanosoma brucei. Western blot analysis of purified organelle fractions revealed the presence of many proteins containing this PTS in both glyoxysomes and glycosomes. These results indicate that at least one of the signals, and therefore the mechanism, for protein translocation into peroxisomes, glyoxysomes, and glycosomes has been conserved, lending support to a common evolutionary origin for these microbodies. Hydrogenosomes, the fourth type of microbody, did not contain proteins that cross-reacted with the anti-PTS antibody, suggesting that this organelle is unrelated to microbodies.     

Peroxisome membrane biogenesis: the stage is set

Pex3p and Pex19p are key players in the post-translational import of peroxisomal membrane proteins. New data suggest that these peroxins act in tandem, Pex19p as a cytosolic chaperone and import receptor for peroxisomal membrane proteins, and Pex3p as docking factor at the peroxisomal membrane.     

Synthesis of novel 4- and 5-substituted benzyl ether derivatives of vesamicol and in vitro evaluation of their binding properties to the vesicular acetylcholine transporter site

Detection of the central cholinergic deficits, a consistent feature of Alzheimer's disease, is essential to allow preventive measures and/or symptomatic treatment already at a very early stage of the disease. The vesicular acetylcholine transporter (VAChT) represents an appropriate target to establish PET radiotracer that are adequate for brain imaging the loss of cholinergic terminals. Here we describe the synthesis and binding characteristics of novel derivatives of vesamicol, known to represent a specific antagonist of VAChT sites. Novel benzyl ether derivatives of vesamicol either 4- or 5-substituted at the cyclohexylring have been synthesized by different regioselective ring opening reactions of a same epoxide precursor. The affinity and selectivity of the novel compounds to VAChT sites were analyzed by competitive radioligand binding studies in rat brain and liver membrane preparations using tritium labeled radioligands. The 4-substituted fluorobenzylether of vesamicol 10b was shown to exhibit a high affinity to VAChT sites (K(i)-value(10b)=10.7+/-1.7 nM), but demonstrated also binding capacities to sigma receptors (K(i-)value(10b)=18.5+/-6.9 nM, [(3)H]DTG; K(i)-value(10b)=30.6+/-9.6 nM, [(3)H]haloperidol). The data suggest the potential of vesamicol derivatives to design appropriate radiotracer for PET imaging of central cholinergic deficits.     

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.     

Glutamate-stimulated secretion of amyloid precursor protein from cortical rat brain slices

The aim of this study was to determine whether L-glutamate, a major excitatory transmitter in the cerebral cortex, modulates the proteolytic cleavage of the amyloid precursor protein (APP) in the brain through specific receptor activation. Native rat brain cerebral cortical slices were stimulated either with L-glutamate or various glutamate receptor agonists, and the soluble APP derivatives released into the incubation medium were assayed by Western blot analysis. Immunoprecipitation with specific antibodies revealed that in the medium only secretory forms of APP lacking intact C-terminus were present, whereas in the brain slices both C- and N-terminal intact APP products were detectable. L-glutamate induced the release of secretory APP from cortical slices in a concentration-dependent but biphasic manner, with the highest release at 50 μM L-glutamate and smaller effects at higher glutamate concentrations. To determine whether the effect of L-glutamate is mediated through distinct glutamate receptor subtypes, brain slices were incubated in the presence of various specific glutamate receptor agonists. Activation of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor with 50 nM (RS)-bromohomoibotenic acid resulted in a reduced release of secretory APP by 17%±3 (P<0.01, one tailed Student's t-test) compared to the incubation without any drug. Stimulation of the metabotropic glutamate receptor with 50 nM (2S,3S,4S)--(carboxycyclopropyl)-glycine (L-CCG-I) led to an enhanced release of secretory APP by 39%±3 (P<0.001), whereas activation of the N-methyl-D-aspartate (NMDA) receptor with 50 nM (1R,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1R,3R)-ACPD) did not significantly change the secretion of APP compared to the incubation without any drug. The data suggest that: (i) cortical glutamatergic neurotransmission is involved in APP metabolism; and (ii) the stimulation of APP cleavage in cerebral cortical brain slices is mainly mediated by the metabotropic but not the NMDA glutamate receptor subtype, whereas the AMPA receptor subtype seems to inhibit the secretory path of APP processing.     

Peroxisomal Targeting of PTS2 Pre-Import Complexes in the Yeast Saccharomyces cerevisiae

Posttranslational matrix protein import into peroxisomes uses either one of the two peroxisomal targeting signals (PTS), PTS1 and PTS2. Unlike the PTS1 receptor Pex5p, the PTS2 receptor Pex7p is necessary but not sufficient to target cargo proteins into the peroxisomal matrix and requires coreceptors. Saccharomyces cerevisiae possesses two coreceptors, Pex18p and Pex21p, with a redundant but not a clearly defined function. To gain further insight into the early events of this import pathway, PTS2 pre-import complexes of S. cerevisiae were isolated and characterized by determination of size and protein composition in wild-type and different mutant strains. Mass spectrometric analysis of the cytosolic PTS2 pre-import complex indicates that Fox3p is the only abundant PTS2 protein under oleate growth conditions. Our data strongly suggest that the formation of the ternary cytosolic PTS2 pre-import complex occurs hierarchically. First, Pex7p recognizes cargo proteins through its PTS2 in the cytosol. In a second step, the coreceptor binds to this complex, and finally, this ternary 150 kDa pre-import complex docks at the peroxisomal membrane, where both the PTS1 and the PTS2 import pathways converge. Gel filtration analysis of membrane-bound subcomplexes suggests that Pex13p provides the initial binding partner at the peroxisomal membrane, whereas Pex14p assembles with Pex18p in high-molecular-weight complexes after or during dissociation of the PTS2 receptor.