Protein kinase Calpha and beta1 isoforms are regulators of alpha-secretory proteolytic processing of amyloid precursor protein in vivo.

We have recently shown that in utero treatment of guinea pigs with the DNA methylating substance methylazoxymethanol acetate (MAM) results in neocortical microencephalopathy, increased protein kinase C (PKC) activity and altered processing of the amyloid precursor protein (APP) in neocortex of offspring. Here we show that PKCalpha and PKCbeta1 are the key regulators of alpha-secretory APP processing in guinea pig neocortex under these experimental conditions in vivo. This conclusion is based on the selective translocation of PKCalpha and PKCbeta1 isoforms to the cell membrane in MAM-treated guinea pigs, as revealed by Western blot analysis and by immunocytochemistry. Additionally, we observed that [3H]phorbol ester binding to protein kinase C increased by 38% and enhanced basal PKC activity by 58% in the neocortex of microencephalic guinea pigs. Inhibition of PKCalpha/PKCbeta1 by Gö6976 abolished this difference, suggesting that constitutive overactivation of these PKC isoforms accounts for the increase in total PKC activity. We also observed a strong positive correlation between levels of alpha-secretase-processed APP and PKC activity in the neocortex of individual animals, providing further evidence for a significant role of classical PKC isoforms in nonamyloidogenic APP processing.

Constitutive overactivation of protein kinase C in guinea pig brain increases alpha-secretory APP processing without decreasing beta-amyloid generation.

Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and beta-amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA-methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester-stimulated PKC enzyme activity (by 47% and 71%) as compared to age-matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol-treated animals, there was increased alpha-secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP alpha-secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of beta-amyloid peptides 1-40 or 1-42 as measured by ELISA, suggesting that only the alpha-secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.

Expression of Alzheimer's disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation.

Numerous mutations causing early onset Alzheimer's disease have been identified in the presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the beta-amyloid precursor protein gene, PS mutations cause the increased generation of a highly neurotoxic variant of amyloid beta-peptide. PS proteins are proteolytically processed to an N-terminal approximately 30-kDa (NTF) and a C-terminal approximately 20-kDa fragment (CTF20) that form a heterodimeric complex. We demonstrate that this complex is resistant to proteolytic degradation, whereas the full-length precursor is rapidly degraded. Degradation of the PS1 holoprotein is sensitive to inhibitors of the proteasome. Formation of a heterodimeric complex is required for the stability of both PS1 fragments, since fragments that do not co-immunoprecipitate with the PS complex are rapidly degraded by the proteasome. Mutant PS fragments not incorporated into the heterodimeric complex lose their pathological activity in abnormal amyloid beta-peptide generation even after inhibition of their proteolytic degradation. The PS1 heterodimeric complex can be attacked by proteinases of the caspase superfamily that generate an approximately 10-kDa proteolytic fragment (CTF10) from CTF20. CTF10 is rapidly degraded most likely by a calpain-like cysteine proteinase. From these data we conclude that PS1 metabolism is highly controlled by multiple proteolytic activities indicating that subtle changes in fragment generation/degradation might be important for Alzheimer's disease-associated pathology.

Proteolytic processing of the Alzheimer disease-associated presenilin-1 generates an in vivo substrate for protein kinase C.

The majority of familial Alzheimer disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). It was shown that the full-length PS-2 protein is phosphorylated constitutively within its N-terminal domain by casein kinases, whereas the PS-1 protein is not. Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of approximately 20-kDa C-terminal fragments (CTF) and approximately 30-kDa N-terminal fragments [Thinakaran, G., et al. (1996) Neuron 17, 181-190]. Here we describe the surprising finding that the CTF of PS-1 is phosphorylated by protein kinase C (PKC). Stimulation of PKC causes a 4- to 5-fold increase of the phosphorylation of the approximately 20-kDa CTF of PS-1 resulting in reduced mobility in SDS gels. PKC-stimulated phosphorylation occurs predominantly on serine residues and can be induced either by direct stimulation of PKC with phorbol-12,13-dibutyrate or by activation of the m1 acetylcholine receptor-signaling pathway with the muscarinic agonist carbachol. However, phosphorylation of full-length PS-1 and PS-2 is not altered upon PKC stimulation. In addition, a mutant form of PS-1 lacking exon 10, which does not undergo endoproteolytic cleavage [Thinakaran, G., et al. (1996) Neuron 17, 181-190] is not phosphorylated by PKC, although it still contains all PKC phosphorylation sites conserved between different species. These results show that PKC phosphorylates the PS-1 CTF. Therefore, endoproteolytic cleavage of full-length PS-1 results in the generation of an in vivo substrate for PKC. The selective phosphorylation of the PS-1 CTF indicates that the physiological and/or pathological properties of the CTF are regulated by PKC activity.

Dependence of cerebrospinal fluid Tau protein levels on apolipoprotein E4 allele frequency in patients with Alzheimer's disease.

Consistent pathological hallmarks of Alzheimer's disease (AD) are the formation of brain amyloid and neurofibrillary tangles (NFTs). Levels of the major protein component of NFTs, the microtubule associated protein Tau, were shown to be increased in cerebrospinal fluid (CSF) of AD patients as compared to age-matched controls. The presence of apolipoprotein E-epsilon 4 allele (APOE4) is a risk factor for sporadic and familial late-onset AD. ApoE may interact with the binding of Tau to microtubules and Tau phosphorylation in an isoform-specific manner. We investigated whether direct evidence of an isoform-specific interaction of apoE and Tau can be demonstrated in the CSF of live AD patients. We measured the apoE genotype and CSF levels of Tau in 19 patients with probable AD and 12 age-matched control subjects. We found that CSF levels of Tau increase with increasing APOE allele frequency (Spearman rank correlation, zeta = 2.71, P = 0.007). This finding may be in agreement with reports of a lesser binding of apoE4 to Tau, compared to apoE2 and apoE3, resulting in higher levels of unbound Tau in CSF.

Muscarinic M1 receptor agonists increase the secretion of the amyloid precursor protein ectodomain.

Amyloid deposits in Alzheimer's disease are composed of amyloid beta-peptides (A beta) that are derived from the larger amyloid precursor protein (APP). Proteolytic APP processing is activity-dependent, and it can be regulated by muscarinic acetylcholine receptors. In particular, muscarinic m1 receptor subtypes increase cleavage within the A beta domain, followed by the release of the soluble APP ectodomain (APPs). In this study, we show that the m1-selective agonist talsaclidine concentration-dependently increased APPs release from both transfected human astrocytoma cell lines and rat brain slices. This increase was blocked by atropine. In contrast, the M2 antagonist BIBN 99 failed to increase APPs release, and decreased it at higher concentrations. These results show that talsaclidine can effectively modulate alpha-secretase processing of APP in human cell lines and in brain tissue. The data suggest that talsaclidine may be a useful candidate drug to modulate APP processing in Alzheimer's disease.

Medicinal chemistry of muscarinic agonists for the treatment of dementia disorders.

Alzheimer disease (AD) is a neurodegenerative disorder lacking an effective therapy. The etiology is controversial and among different drug strategies, the cholinergic approach has gained great interest owing to biochemical and pharmacological evidence of the crucial role of acetylcholine in cognitive functions. Several attempts exploiting the boosting of the cholinergic system are currently under way. Inhibitors of the acetylcholinesterase enzyme sustain the availability of the natural transmitter by limiting its removal from the synapse. In a different approach, exogenous agonists may substitute acetylcholine itself. In this way the issue of the extensive cholinergic cell loss occurring in AD and leading to a reduction of cholinergic functions, could be advantageously bypassed. Moreover the discovery of different muscarinic receptor subtypes, most notably the M1 subtype as that involved in the postsynaptic transmission, has offered new opportunities to face the problem in a very specific way. In this line of research, we have now identified BIMC 182 as a new functionally selective M1 agonist. Whereas its affinity for the different receptor subtypes is almost similar (radioreceptor binding), its functional selectivity is pointed out by specific "in vitro" models. BIMC 182 behaves as a full agonist at M1 (rat superior cervical ganglion, pD2 4.8) and as a partial agonist at M2 and M3 sites (g.p. heart pD2 = 5.4 and g.p. ileum pD2 = 4.5). The agonist profile is further confirmed in hm1 transfected CHO cells where the compound stimulates PI turnover. BIMC 182 penetrates well the brain as shown by the increase in the energy of the low frequency band (theta waves) in the cortical EEG of rabbits (3 mg/kg i.v.).(ABSTRACT TRUNCATED AT 250 WORDS)

WAL 2014--a muscarinic agonist with preferential neuron-stimulating properties.

The ability of WAL 2014 to elicit muscarinic responses was investigated in various in vitro and in vivo models. In CHO cells transfected with human m1- or m3- receptor genes, WAL 2014 was clearly more effective in stimulating the M1-mediated PI response. In isolated tissue preparations, WAL 2014 exhibited full agonist properties in the rabbit vas deferens (putative M1 receptor) and behaved like a partial agonist at M2 receptors in the atrium and M3 receptors in the ileum of guinea-pigs. In the pithed rat, in which the increase in blood pressure is mediated through a stimulation of M1 receptors in sympathetic ganglia, WAL 2014 produced a full dose response curve, whereas the reference compounds RS 86 and arecoline exhibited a bell-shaped behaviour. This is in accord with the view that WAL 2014 selectively activates M1 receptors in sympathetic ganglia, whereas conventional agonists in the same dose range stimulate both ganglionic M1 and vascular M3 receptors. The preferential neuron-stimulating properties were confirmed by EEG recording in the rabbit, in which muscarinic activation occurred at doses similar to those for ganglionic stimulation in the pithed rat. On the other hand, higher doses of WAL 2014 were needed to elicit muscarinic effects in peripheral effector organs, i.e. bradycardia, urinary bladder contraction and increase in airway resistance. It is concluded that WAL 2014 due to its preferential neuronal activity is a promising candidate for a cholinergic substitution therapy in Alzheimer's disease.

Cloning and functional characterization of a human A1 adenosine receptor.

A human brain hippocampus cDNA library was screened by hybridization with a dog A1 adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog A1 adenosine receptor. Individual clones of stably transfected CHO cells expressing the human A1 receptor were obtained and tested for their response to the A1 agonist CPA [N6-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an A1 adenosine receptor. A Kd value of 2.28 nM was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an A1 selective antagonist.

Defective lysosomal release of glycoprotein-derived sialic acid in fibroblasts from patients with sialic acid storage disease.

Fibroblasts from patients with sialic acid storage disease (SASD), sialidosis, mucolipidosis II, and from normal controls, were incubated in the presence of the glycoprotein fetuin that was tritium-labelled in its sialic acid residues by the periodate/[3H]borohydride reduction method, and the fate of the intracellular radioactive sialic acid (C7-sialic acid) followed in pulse-chase experiments. The model glycoprotein was readily endocytosed and degraded, more than 90% of the radioactivity being trichloroacetic acid (TCA)-soluble after 4 days of incubation. In all of the patients' fibroblasts, there was an increased accumulation of TCA-soluble radioactivity and, upon chase, a much lower rate of elimination than in normal controls. Gel chromatography of the material from the chase experiment showed that, in normal cells, most of the radioactivity at zero time behaved as free C7-sialic acid. This, as well as material of larger size (sialyloligosaccharides), was very much diminished by 48 h. In cells from two patients with SASD, there were large peaks both in the sialic acid and oligosaccharide positions; whereas the oligosaccharides were somewhat decreased by the end of the chase period, the sialic acid was essentially unchanged. In sialidosis fibroblasts, the radioactive material consisted of oligosaccharides, but very little C7-sialic acid; the elimination of the oligosaccharides was retarded. In normal cells, about 80% of the radioactivity released into the medium after 48 h chase behaved as free C7-sialic acid upon gel chromatography and t.l.c. Subcellular fractionation in Percoll gradients showed that the radioactive C7-sialic acid remaining in normal cells after 48 h of chase was mainly localized in the cytosol. In SASD cells, on the other hand, it was associated with lysosomal fractions which, unexpectedly, exhibited an abnormally low density. Our findings demonstrate that SASD fibroblasts degrade the sialoglycoprotein but, unlike normal cells, accumulate the liberated C7-sialic acid along with sialyloligosaccharides in their lysosomes. The results therefore support the concept of a defective transport system for sialic acid in the lysosomal membrane of patients with SASD.

Prenatal diagnosis and confirmation of infantile sialic acid storage disease.

Amniocentesis was performed in a pregnancy at risk for infantile sialic acid storage disease. Greatly elevated levels of free sialic acid were found in cell-free amniotic fluid as well as in cultured amniotic cells from the fetus at risk. After incubation of the cultured amniocytes with fetuin labelled in its sialic acid moiety, pulse and chase experiments respectively showed accumulation and impaired release of TCA-soluble radioactive material in the amniotic cells at risk. These data thus clearly indicated that the fetus was affected. After pregnancy termination, ultrastructural studies of fetal organs and placenta showed a generalized storage picture characterized by clear membrane-bound inclusions. The diagnosis was further confirmed by the finding of greatly increased amounts of free sialic acid in fetal organs and cultured fibroblasts.

N-Acetylneuraminic acid storage disease.

Increased amounts of free sialic acid were found in body fluids, leukocytes, cultured fibroblasts, and liver tissue of a four-year-old boy with mental retardation, ataxia, and clinical and radiologic findings of a mild mucopolysaccharidosis. A diagnosis of Salla disease was made though in contrast to earlier reports, recurrent upper respiratory infections and hepatosplenomegaly were present already in infancy, and skeletal abnormalities of dysostosis multiplex were found in early childhood. Free sialic acid in the urine was identified as N-acetylneuraminic acid by 1H-NMR spectroscopy. Sialidase activities were normal. Increased amounts of bound sialic acid were found in liver and cultured fibroblasts and were attributed to an intracellular inhibition of sialyloligosaccharide-degrading neuraminidase by excessive amounts of free neuraminic acid. The molecular basis of N-acetylneuraminic acid storage disease is unknown but may be related to a defective transport mechanism preventing neuraminic acid from leaving the lysosomal compartment.

Partial deficiency of glycoprotein neuraminidase in some patients with Morquio disease type A.

A deficiency of glycoprotein neuraminidase (sialidase, acylneuraminyl hydrolase, EC 3.2.1.18) activity was found in fibroblasts from a patient with the clinical symptoms of Morquio disease type A (mucopolysaccharidosis IV A). Residual neuraminidase activity was about 5% of the mean normal activity. N-Acetylgalactosamine-6-sulfate (GalNAc-6-S) sulfatase activity was reduced to less than 1% of normal with a pH-optimum of 3.0 as expected for the severe form of Morquio disease. In peripheral leucocytes of the patient, however, neuraminidase activity but not Ga1NAc-6-S sulfatase activity was in the normal range. Mixing experiments excluded the presence of excessive amounts of inhibitors of neuraminidase activity.

Specificity studies on the oligosaccharide neuraminidase of human fibroblasts.

Competition and thermal inactivation experiments with different potential natural substrates indicated that in homogenates of human fibroblasts one single enzyme is acting on both (alpha 2-3) and (alpha 2-6) sialosyl linkages of oligosaccharides and glycoproteins, but not of the ganglioside GM3. N-Acetylneuraminic and 2-deoxy-2,3-dehydro-N-acetylneuraminic acids are competitive inhibitors, whereas chondroitin 4-sulphate and the drug Suramin are potent inhibitors of undefined type.