Jonathan Turner

Generation of βA4 from the amyloid protein precursor and fragments thereof

The cellular mechanisms underlying the generation of βA4 in Alzheimers disease and its relationship to the normal metabolism of the amyloid protein precursor (APP) are unknown. In this report, we show that expression of the C‐terminal 100 residues of APP, with (SPA4CT) or without (A4CT) a signal sequence in the N‐terminal position, in human neuroblastoma cells results in secretion of a 4 kDa βA4‐like peptide. In A4CT and SPA4CT expressing SY5Y cells, βA4 generation could not be inhibited by the lysosomotropic amines chloroquine and ammonium chloride but was inhibited by brefeldin A, monensin and methylamine. The last also selectively inhibits APP secretion in neuroblastoma cells [1]. The finding that chloroquine and ammonium chloride inhibit βA4 generation from full length APP but not from A4CT and SPA4CT are consistent with the assumption that the two cleavages necessary to generate βA4 operate in two different compartments. Our data suggest the cleavage which generates the C‐terminus of βA4 takes place in the same compartment (late Golgi or endosomal vesicles) in which the APP‐secretase operates.

Amyloid precursor protein secretion and βA4 amyloid generation are not mutually exclusive

The cellular factors regulating the generation of βA4 from the amyloid precursor protein (APP) are unknown. Protein phosphorylation by protein kinase C (PKC) has been found to influence the processing and metabolism of APP. In this report, we show that in the human neuroblastoma cell line SY5Y, βA4 generation from full‐length APP is not changed by PKC activation whereas production of the non‐amyloidogenic secretory fragment (APPsec) and of the C‐terminal fragment of βA4 (p3) are stimulated. In addition, βA4 generation from the membrane inserted C‐terminal 100 residues (SPA4CT) of APP is stimulated by PKC activation. Accordingly attempts to divert APP processing from the amyloidogenic, βA4‐generating, to the non‐amyloidogenic, secretory, pathway, have to address the nature and regulation of the two pathways and/or of the process leading to the cleavage of APP at the C‐terminus of the βA4 domain. The data reported here suggest that these mechanisms are cell‐type specific.

Mapping of GABAA receptor α5 and α6 subunit-like immunoreactivity in rat brain

The distribution of the α5 and α6 subunits of the GABAA receptor has been mapped in rat brain using affinity-purified antibodies generated against peptide sequences unique to the respective polypeptides. α5 Subunit-like immunoreactivity was of low density but was distributed across several cell groups including cortical interneurones, hippocampal CA3 pyramidal neurones, the anterior thalamic reticular nucleus and cerebellar Purkinje neurones. α6 Subunit-like immunoreactivity was observed in high density in cerebellar granule cells. These patterns are compatible with in situ hybridisation studies and provide a further anatomical substrate for GABAA receptor heterogeneity in the CNS.

5-HT1A receptor agonists improve the performance of normal and scopolamine-impaired rats in an operant delayed matching to position task

A series of experiments examined the effects of 5-HT1A ligands alone and in combination with the muscarinic antagonist scopolamine on short term working memory in the rat. The behavioural paradigm was a discrete trial, operant delayed matching to position task, with delays of 0, 5, 15 and 30 s. The 5-HT1A ligands tested were the full agonist, 8-OH DPAT (0, 0.1, 0.3 and 1 mg/kg), the partial agonist, ipsapirone (0, 1, 3 and 10 mg/kg), and the purported antagonist, NAN 190 (0, 1, 2, and 4 mg/kg). 1-PP (0, 0.1, 0.3, 1 mg/kg), the major metabolite of ipsapirone, was also tested. The lowest dose of 8-OH DPAT significantly improved matching accuracy at the longest delay, whereas the highest dose impaired matching accuracy and increased the latency to respond. Ipsapirone also significantly improved the accuracy of performance at a dose of 3 mg/kg, but the doses of 1 and 10 mg/kg did not significantly affect performance. NAN-190, at the highest dose tested (4 mg/kg), impaired matching accuracy, whereas the two lower doses did not significantly affect performance. The highest dose also increased the latency to respond. 1-PP had no effect on performance. Scopolamine HBr (0.14 mg/kg) caused a delay dependent impairment in matching accuracy, and had no effect on missed trials or the latency to respond. Low doses of 8-OH DPAT (0.1 and 0.3 mg/kg) significantly attenuated the scopolamine induced accuracy impairment, whereas 1 mg/kg 8-OH DPAT potentiated the impairment. Ipsapirone (3 mg/kg) also significantly improved the performance of scopolamine impaired rats. NAN-190 increased the latency to respond and reduced the number of nose pokes made during the delays in scopolamine-treated rats, and tended to potentiate the scopolamine-induced accuracy impairment. 1-PP did not affect the performance of scopolamine treated rats. Taken together, these results suggest that modulation of 5-HT1A receptors influences short term spatial working memory in the rat.

Effects of NBM lesions with two neurotoxins on spatial memory and autoshaping

Four groups of Wistar rats received either vehicle, quisqualate, or one of two different ibotenic acid infusions into the basal forebrain. Following recovery from surgery, all rats were tested in three distinct behavioral paradigms: the Bättig radial arm maze, the Barnes circular platform, and autoshaping in an operant chamber. The results showed that the size and site of the ibotenic acid lesion had a profound effect on acquisition performance in some, but not all, procedures. Performance in the Bättig maze and acquisition of a food-rewarded lever press were in particular disrupted by ibotenic acid lesions. The severity of the reduction in cortical choline acetyltransferase (ChAT) did not correlate with performance in the tests. Quisqualate produced the largest reduction in ChAT levels but had no significant effect on performance in any of the three procedures used. Anatomic analysis revealed severe nonspecific damage to the striatum following ibotenic acid that was more pronounced in the group receiving a highly concentrated solution of ibotenic acid as compared to rats infused with a greater volume but less concentrated solution of the neurotoxin. Striatal damage was much less severe following quisqualic acid infusions. However, both types of neurotoxins produced equivalent nonspecific degeneration of the reticular thalamic nucleus. These data confirm reports that nonspecific damage appears to define the severity of ibotenic acid lesions on subsequent behavioral performance.

Co-localization of β-amyloid peptides, apolipoprotein E and glial markers in senile plaques in the prefrontal cortex of old rhesus monkeys

Based on the homology of human and monkey amyloid precursor proteins and the derived beta-amyloid peptides (A beta) the investigation of brains from old monkeys might be useful for the understanding of beta-amyloidosis in the aetiology of Alzheimers disease. In the present study, the prefrontal cortex, which is known to be highly susceptible to the deposition of A beta, was screened for the occurrence of senile plaques in perfused tissue of aged rhesus monkeys (Macaca mulatta). A beta deposits were immunocytochemically detected in five of six macaques aged about 28 years. Differently N-terminal truncated A beta species in the senile plaques were simultaneously detected by a carbocyanine double fluorescence method applying the bright red fluorescent Cy3 and the novel green fluorescent Cy2. In a few cases, immunoreactivity for the shortened fragment containing the amino acids 17-42 (A beta(17-42); p3 fragment with a molecular weight of 3 kDa) was demonstrated in deposits apparently devoid of A beta(8-17). Senile plaques were further characterized by carbocyanine double labelling of A beta and astrocytes, microglia and apolipoprotein E.

Immunohistochemical Mapping of Gamma-Aminobutyric Acid Type-A Receptor Alpha Subunits in Rat Central Nervous System

The inhibitory γ-aminobutyric acid type A (GABAA) receptors of mammalian brain are hetero-oligomeric membrane glycoproteins, in which five subunits are thought to assemble to form individual chloride-channel complexes bearing GABAA receptors and their associated modulatory sites. Five distinct subunit classes, some containing several isoforms (α1–6, β1–3, γ1–3, 5 and ρ1–2) encoded by separate genes, have been identified by molecular cloning (see e.g., Olsen and Tobin 1990; Stephenson 1991). Recombinant receptors assembled from these subunits possess heterogeneous pharmacological and biophysical properties, with the pharmacological properties of the principal allosteric modulatory site (the benzodiazepine receptor, BZR) being determined by the α and/or γ species present (see e.g., Luddens and Wisden 1991). The combinations which assemble in vivo to constitute native receptors are not known, but in situ hybridisation studies have demonstrated not only overlapping but also distinct distributions of the mRNAs coding for the different subunit classes and isoforms (e.g., Laurie et al. 1992; Wisden et al. 1992). While ligand-binding studies in brain membranes employing compounds such as Zolpidem (Niddam et al. 1987) have previously provided evidence for the existence of two classes of BZR (termed BZ1 and BZ2 or ω1 and ω2), this recent evidence from molecular biological studies suggests the presence of subclasses at least of the BZ2 type (e.g., Pritchett and Seeburg 1990).

Cationic Lipids (Lipofectamine) and Disturbance of Cellular Cholesterol and Sphingomyelin Distribution Modulates Gamma-Secretase Activity Within Amyloid Precursor Protein In Vitro

To study beta-amyloid protein generation we expressed different amyloid precursor protein (APP) isoforms in the human neuroblastoma cell line SY5Y (for details see (1)). Treatment with lipofectamine, an cationic lipid for eucaryotic cell transfection, inhibits gamma-secretase activity and stimulates the physiological APP cleavage by alpha-secretase activity. Beside the MDL inhibitor (2), this is the second agent that shows modulation of gamma-secretase activity in vitro. Further, we show that disturbance of cellular cholesterol and sphingomyelin distribution in transfected SY5Y cells results in an overproduction of beta-amyloid protein. This provides experimental evidence that membrane instability influenced the proteolytic activity of gamma-secretase within the APP molecule.

Pulse-chase experiments revealed β-secretase cleavage from immature full-length amyloid precursor protein harboring the swedish mutation

The molecular mechanisms of the nonamyloidogenic and the amyloidogenic pathways of the amyloid precursor protein (APP) are unknown, but proteolysis of APP is essential for the generation of β-amyloid. To study the time-course of C-terminal fragment generation by α- and β-secretase, we expressed the APP751 isoform with the Swedish mutation in the human neuroblastoma cell line SY5Y as previously described (Urmoneit et al., 1995). We show in pulse-chase experiments that the C-terminal fragments, CT, generated by α-secretase and A4CT, generated by β-secretase, could be generated from immature full-length APP before O-glycosylation is completed. Thus βA4 may be generated from immature APP that has not passed through the trans-Golgi-network (TGN), which presents experimental evidence for the intracellular localization of β-secretase activity in an earlier Golgi complex.

Inhibition of βA4 production by specific modulation of β-secretase activity

To study amyloid precursor protein (APP) processing we expressed different APP isoforms with and without the Swedish mutation and the membrane inserted C-terminal 100 residues of APP (SPA4CT) in the human neuroblastoma cell line SY5Y. We show that expression of the Swedish mutation results in a significant production of the amyloidogenic intermediate A4CT, which is further processed by γ-secretase leading to an overproduction of βA4. Treatment with methylamine and ammonium chloride, inhibitors interfering with intracellular transport mechanisms, inhibits β-secretase activity without influencing the physiological APP cleavage by α-secretase activity. By expressing SPA4CT, we demonstrate that secretion, but not generation, of βA4 from SPA4CT is inhibited by methylamine resulting in intracellular βA4. This provides experimental evidence for the intracellular localization of γ-secretase activity and βA4 generation.