Non-amyloidogenic Amyloid Precursor Protein Research Articles

Cholinesterase inhibitors influence APP metabolism in Alzheimer disease patients

Platelets mirror pathogenic alterations in the central nervous system of Alzheimer disease (AD) patients: an alteration of the Amyloid Precursor Protein (APP) forms pattern and decreased alpha-secretase activity--the non-amyloidogenic APP processing enzyme--were demonstrated. Platelets were analysed at baseline and after 30 days of cholinesterase inhibitor (ChEI) treatment (T30). ADAM10 levels, alpha- and beta-secretase activity were assessed measuring ADAM10 immunoreactivity, sAPPalpha release and the membrane-attached C-terminal fragments produced by beta- and alpha-secretase cleavage, that is, CTF99 and CTF83, respectively. ChEIs treatment rescues impaired APP metabolism increasing significantly ADAM10 levels (T30 vs. T0, P < 0.05), alpha-secretase activity (T30 vs. T0, P < 0.05) and reducing beta-secretase cleavage (T30 vs. T0, P < 0.05). Restoration of the balance between the mutually exclusive alpha- and beta-secretase pathway in APP processing caused by short-term ChEIs treatment potentially represents a key event in AD therapy linking in vivo cholinergic effect to APP metabolism. The use of platelets may represent a useful tool to follow molecular aspects of pharmacological response in AD patients.

The anti-inflammatory and cholinesterase inhibitor bifunctional compound IBU-PO protects from β-amyloid neurotoxicity by acting on Wnt signaling components

Changes in signal transduction are implicated in neuronal responses to the Alzheimer's amyloid-beta-peptide (Abeta), which include neurotransmitter systems and pathways involved in the maintenance of the nervous system. We report here that a new bifunctional compound IBU-PO, which combines a non-steroidal anti-inflammatory drug (NSAID) (Ibuprofen) and a cholinesterase (ChE) inhibitor (Octyl-Pyridostigmine), is neuroprotective against Abeta-neurotoxicity, and its activity is associated to Wnt signaling components in rat hippocampal and mouse cortical neurons. IBU-PO (0.01-1 microM) inhibits glycogen-synthase-kinase-3beta (GSK-3beta) and stabilizes cytoplasmic beta-catenin reverting the silencing of the Wnt pathway caused by Abeta-toxicity and GSK-3beta overexpression. In addition, IBU-PO enhances, dose-dependently, the non-amyloidogenic amyloid precursor protein (APP) cleavage by increasing secreted APP and decreasing endogenous Abeta1-40 in rat hippocampal neurons.

Controlling neural stem cell division within the adult subventricular zone: an APPealing job

For years, scientists investigating amyloid precursor protein (APP) have focused on its pathogenetic role in the brains of Alzheimer's disease patients. Now, a study by Caille et al. adds new sites of action and new physiological functions for APP. They show that there are binding sites for secreted N-terminal nonamyloidogenic APP (sAPP) on epidermal growth factor (EGF)-responsive neural stem cells in the subventricular zone of the adult brain, where sAPP acts as an EGF cofactor to stimulate proliferation of these cells. This result opens the hypothesis that changes in the levels of sAPP could influence activity of the neurogenic regions of the adult brain in normal and pathological conditions.

Acetylcholinesterase and its inhibition in Alzheimer disease.

Until recently, the only established function of acetylcholinesterase (AChE) was the termination of cholinergic neurotransmission. Therefore, the use of AChE inhibitors to treat symptoms caused by cholinergic imbalances in Alzheimer disease (AD) represented a rational approach. However, it is now clear that AChE and the cholinergic system may have broader effects in AD. Of particular interest may be signal transduction pathways mediated through cholinergic receptors that promote nonamyloidogenic amyloid precursor protein processing and decrease tau phosphorylation, and the role of AChE in the aggregation of beta-amyloid (Abeta) peptide. In addition, the neuronal and nonneuronal cholinergic systems have important roles in the modulation of regional cerebral blood flow. These findings may modify the overly simplistic cholinergic hypothesis in AD that is limited to symptomatic treatment and ignores the potential of cholinergic therapies as disease-modifying agents. Chronic increases in AChE activity may exacerbate neurodegenerative processes, make clinically relevant levels of AChE inhibition more difficult to achieve, and cause the therapeutic value of cholinesterase inhibitors (ChE-Is) to be limited and temporary. Rapidly reversible ChE-Is appear to increase AChE activity over the longer term whereas, remarkably, irreversible or very slowly reversible ChE-Is do not seem to have this effect. If such differences between ChE-Is are shown to have clinical correlates, this may prompt reconsideration of the rationale and expectations of some agents in the long-term management of AD.

Neuroprotection and neurorescue against Abeta toxicity and PKC-dependent release of nonamyloidogenic soluble precursor protein by green tea polyphenol (-)-epigallocatechin-3-gallate.

Green tea extract and its main polyphenol constituent (-)-epigallocatechin-3-gallate (EGCG) possess potent neuroprotective activity in cell culture and mice model of Parkinson's disease. The central hypothesis guiding this study is that EGCG may play an important role in amyloid precursor protein (APP) secretion and protection against toxicity induced by beta-amyloid (Abeta). The present study shows that EGCG enhances (approximately 6-fold) the release of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPalpha) into the conditioned media of human SH-SY5Y neuroblastoma and rat pheochromocytoma PC12 cells. sAPPalpha release was blocked by the hydroxamic acid-based metalloprotease inhibitor Ro31-9790, which indicated mediation via alpha-secretase activity. Inhibition of protein kinase C (PKC) with the inhibitor GF109203X, or by down-regulation of PKC, blocked the EGCG-induced sAPPalpha secretion, suggesting the involvement of PKC. Indeed, EGCG induced the phosphorylation of PKC, thus identifying a novel PKC-dependent mechanism of EGCG action by activation of the non-amyloidogenic pathway. EGCG is not only able to protect, but it can rescue PC12 cells against the beta-amyloid (Abeta) toxicity in a dose-dependent manner. In addition, administration of EGCG (2 mg/kg) to mice for 7 or 14 days significantly decreased membrane-bound holoprotein APP levels, with a concomitant increase in sAPPalpha levels in the hippocampus. Consistently, EGCG markedly increased PKCalpha and PKC in the membrane and the cytosolic fractions of mice hippocampus. Thus, EGCG has protective effects against Abeta-induced neurotoxicity and regulates secretory processing of non-amyloidogenic APP via PKC pathway.

Estrogen-induced cell signalling in a cellular model of Alzheimer’s disease

Alzheimer’s disease (AD) is characterised by deposition of a 4 kDa amyloid-β peptide (Aβ) into senile plaques of the affected brain. Aβ is a proteolytic product of the membrane protein, amyloid precursor protein (APP). An alternative cleavage pathway involves α-secretase activity and results in secretion of a 100 kDa non-amyloidogenic APP (sAPPα) and therefore a potential reduction in Aβ secretion. We have shown that estrogen induces α-cleavage and therefore results in the secretion of sAPPα. This secretion is signalled via MAP-kinase and PI-3 kinase signal-transduction pathways. These pathways also have the potential to inhibit the activation of glycogen synthase kinase 3β (GSK), a protein involved in cell death. Therefore, the aim of this work was to further elucidate the estrogen-mediated signaling pathways involved in APP processing, with particular emphasis on GSK activity. By stimulating rat hypothalamic neuronal GT1-7 cells with estradiol, we found that estrogen decreases the activation state of GSK via the MAP kinase pathway. Moreover, the inhibition of GSK activity by LiCl causes enhanced sAPPα secretion in a pattern similar to that seen in response to estrogen, suggesting a pivotal role for this deactivation in APP processing. Further, inactivation of GSK by estrogen can be confirmed in an in vivo model. Elucidation of the signaling pathways involved in APP processing may help to understand the pathology of AD and may also prove beneficial in developing therapeutic strategies to combat AD.

AF150(S) and AF267B: M1 muscarinic agonists as innovative therapies for Alzheimer's disease.

The M1 muscarinic agonists AF102B (Cevimeline, EVOXACTM: prescribed in USA and Japan for Sjogren's Syndrome), AF150(S) and AF267B--1) are neurotrophic and synergistic with neurotrophins such as nerve growth factor and epidermal growth factor; 2) elevate the non-amyloidogenic amyloid precursor protein (alpha-APPs) in vitro and decrease beta-amyloid (A beta) levels in vitro and in vivo; and 3) inhibit A beta- and oxidative-stress-induced cell death and apoptosis in PC12 cells transfected with the M1 muscarinic receptor. These effects can be combined with the beneficial effects of these compounds on some other major hallmarks of Alzheimer's disease (AD) (e.g. tau hyperphosphorylation and paired helical filaments [PHF]; and loss of cholinergic function conducive to cognitive impairments.) These drugs restored cognitive impairments in several animal models for AD, mimicking different aspects of AD, with a high safety margin (e.g. AF150[S] >1500 and AF267B >4500). Notably, these compounds show a high bioavailability and a remarkable preference for the brain vs. plasma following p.o. administration. In mice with small hippocampi, unlike rivastigmine and nicotine, AF150(S) and AF267B restored cognitive impairments also on escape latency in a Morris water maze paradigm in reversal learning. Furthermore, in aged and cognitively impaired microcebes (a natural animal model that mimics AD pathology and cognitive impairments), prolonged treatment with AF150(S) restored cognitive and behavioral impairments and decreased tau hyperphosphorylation, PHF and astrogliosis. Our M1 agonists, alone or in polypharmacy, may present a unique therapy in AD due to their beneficial effects on major hallmarks of AD.

The Human Serotonin 5-HT4 Receptor Regulates Secretion of Non-amyloidogenic Precursor Protein

The serotonin 5-HT(4) receptor has recently gained a lot of attention for its functional roles in central processes such as memory and cognition. In this study, we show that activation of the human 5-HT(4) (h5-HT(4)) receptor stimulates the secretion of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPalpha). 5-HT enhanced the level of secreted sAPPalpha in a time- and dose-dependent manner in Chinese hamster ovary cells stably expressing the h5-HT(4(e)) receptor isoform. The increase was inhibited by the selective 5-HT(4) receptor antagonist, GR113808. The 5-HT(4) selective agonists, prucalopride and renzapride, also increased secreted sAPPalpha in IMR32 human neuroblastoma cells. The stimulatory effect of 5-HT was mimicked by forskolin, a direct activator of adenylyl cyclase, and 8-bromo-cAMP, a membrane-permeant cAMP analogue. On the contrary, inhibition of protein kinase A (PKA) by H89 potentiated the 5-HT-induced increase in both secreted and cellular sAPPalpha. This phenomenon involves a novel PKA-independent stimulatory process that overcomes a PKA-dependent inhibitory one. Finally, activation of the h5-HT(4(e)) receptor did not modify extracellular amyloid beta-protein in Chinese hamster ovary cells transfected with the human APP695. Given the neuroprotective and enhancing memory effects of sAPPalpha, our results may open a new avenue for the treatment of Alzheimer's disease.

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.

Regulation of APP Synthesis and Secretion by Neuroimmunophilin Ligands and Cyclooxygenase Inhibitors

We and others previously showed that both the synthesis of the amyloid precursor protein (APP) and its processing (i.e., to amyloidogenic A beta peptides; soluble nonamyloidogenic APPs; and other APP fragments) are regulated by neurotransmitters. Transmitters that elevate cellular cAMP levels (like norepinephrine and prostaglandins, which act on beta-adrenergic receptors and prostaglandin E2 receptors respectively) enhance APP synthesis and the formation of amyloidogenic APP holoprotein. Transmitters that stimulate phosphatidylinositol hydrolysis (by activating muscarinic m1 or m3 receptors, serotoninergic 5HT2a or 5HT2c receptors, or metabotropic glutamate receptors of subtypes 1 or 5) increase the conversion of APP to soluble APPs, and decrease the formation of A beta. These findings suggest that drugs that regulate the activity of neurotransmitter receptors might be useful in preventing the excessive formation of A beta or other amyloid precursors in Alzheimer's disease. We now show that neuroimmunophilin ligands (like cyclosporin A or FK-506) and nonsteroidal antiinflammatory agents (NSAIDs), including cyclooxygenase (COX)-2 inhibitors, can also prevent APP overexpression and the overproduction of amyloidogenic peptides. We observe that the enhancement of APP overexpression by prostaglandin E2 is inhibited by neuroimmunophilin ligands like cyclosporin A or FK-506 (tacrolimus). We also find that the NSAIDs, which reduce prostaglandin synthesis by inhibiting COX-1 and -2 enzymes, might also be expected to lower APP levels. Our present data confirm that these drugs, as well as drugs that selectively inhibit COX-2, reduce the levels of amyloidogenic APP holoprotein in cultured neurons or in cultured astrocytes. We previously showed that elevations in cAMP, perhaps generated in response to prostaglandins, can suppress APPs secretion. The NSAIDs and COX inhibitors also increased levels of soluble APPs in the media of cultured astrocytes and neurons, perhaps acting by inhibition of prostaglandin production. Since APP holoprotein can be amyloidogenic, while APPs may be neurotrophic, our findings suggest that some neuroimmunophilin ligands, NSAIDs and COX-2 inhibitors might suppress amyloid formation and enhance neuronal regeneration in Alzheimer's disease.

Brain plasmin enhances APP α‐cleavage and Aβ degradation and is reduced in Alzheimer's disease brains

The proteolytic processing of amyloid precursor protein (APP) has been linked to sphingolipid-cholesterol microdomains (rafts). However, the raft proteases that may be involved in APP cleavage have not yet been identified. In this work we present evidence that the protease plasmin is restricted to rafts of cultured hippocampal neurons. We also show that plasmin increases the processing of human APP preferentially at the alpha-cleavage site, and efficiently degrades secreted amyloidogenic and non-amyloidogenic APP fragments. These results suggest that brain plasmin plays a preventive role in APP amyloidogenesis. Consistently, we show that brain tissue from Alzheimer's disease patients contains reduced levels of plasmin, implying that plasmin downregulation may cause amyloid plaque deposition accompanying sporadic Alzheimer's disease.

Regulation of Amyloid Precursor Protein (APP) Secretion by Protein Kinase Cα in Human Ntera 2 Neurons (NT2N)

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic APP products. Protein kinase C (PKC) has been shown to regulate APPs secretion, and PKCalpha and PKCepsilon have been implicated in APPs secretion in fibroblasts. This study examined the PKC isoform involved in regulated APPs secretion in human NT2N neurons and in CHO cells stably expressing APP(695). Inhibition of PMA-induced APPs secretion with the PKC inhibitors Calphostin C and GF109203X demonstrated that PKC is involved in PMA-regulated APPs secretion in NT2N cells. The specific PKC isoforms present in NT2N and CHO695 cells were identified, and PKCalpha and PKCepsilon were found to translocate from cytosol to membranes in NT2N and CHO695 cells. Translocation of PKC to the membrane allows for activation of the enzyme, as well as for positioning of the enzyme close to its substrate. Long-term PMA treatment led to complete downregulation of PKCalpha in NT2N cells and to downregulation of PKCalpha and PKCepsilon in CHO695 cells. PKCalpha downregulation in the NT2N cells resulted in loss of PMA-regulated APPs secretion and a substantial reduction in constitutive APPs secretion. Downregulation of PKCalpha and PKCepsilon in CHO695 cells resulted in loss of PMA-regulated APPs secretion; however, constitutive APPs secretion was unaffected. These findings suggest that PKCalpha is involved in PMA-regulated APPs secretion in NT2N cells and PKCalpha and/or PKCepsilon is involved in PMA-regulated APPs secretion in CHO695 cells.

Looking for drug targets for the treatment of neurological disease — a focus on lipid and cholesterol pathways

Amyloid-β (Aβ) oligomers have been accepted as major neurotoxic agents in the therapy of Alzheimer’s disease (AD). It has been shown that the activity of nicotinamide adenine dinucleotide (NAD+) is related with the decline of Aβ toxicity in AD. Nicotinamide mononucleotide (NMN), the important precursor of NAD+, is produced during the reaction of nicotinamide phosphoribosyl transferase (Nampt). This study aimed to figure out the potential therapeutic effects of NMN and its underlying mechanisms in APPswe/PS1dE9 (AD-Tg) mice. We found that NMN gave rise to a substantial improvement in behavioral measures of cognitive impairments compared to control AD-Tg mice. In addition, NMN treatment significantly decreased β-amyloid production, amyloid plaque burden, synaptic loss, and inflammatory responses in transgenic animals. Mechanistically, NMN effectively controlled JNK activation. Furthermore, NMN potently progressed nonamyloidogenic amyloid precursor protein (APP) and suppressed amyloidogenic APP by mediating the expression of APP cleavage secretase in AD-Tg mice. Based on our findings, it was suggested that NMN substantially decreases multiple AD-associated pathological characteristically at least partially by the inhibition of JNK activation.

M1 muscarinic agonists: Their potential in treatment and as disease-modifying agents in Alzheimer's disease

M1-type muscarinic receptors (mAChR) have an important role in cognitive processing and are relatively unchanged in Alzheimer's disease (AD). Therefore, M1 agonists represent a rational treatment strategy of AD. However, some muscarinic agonists showed disappointing results in Phase III studies in AD patients. These agonists lacked M1 selectivity in vivo and/or had several major clinical limitations, precluding a proper testing of the clinical concept. There is now justified hope that selective M1 agonists could provide limited causal therapy in AD. Thus, a relation between the formation of β-amyloid peptide and amyloid plaques, tau phosphorylation, and loss of cholinergic function in AD brains has been reported. This may shift the interest in such compounds from a mere symptomatic treatment toward their use in the future as disease-modifying agents via muscarinic regulation of β-amyloid metabolism and tau phosphorylation. Such characteristics can be detected in the functionally selective M1 agonists from the AF series (e.g. AF102B, AF150(S), AF267B). These M1 agonists, inter alia, restore cognitive impairments in several animal models for AD, promote the neurotrophic and nonamyloidogenic amyloid precursor protein (APPs) processing pathways, and decrease tau protein phosphorylation. Apolipoprotein E-deficient mice have memory deficits, synaptic loss of basal forebrain cholinergic projections, and hyperphosphorylation of distinct epitopes of the microtubule-associated protein tau. These impairments are restored by subchronic treatment with AF150(S). Furthermore, prolonged administration of AF150(S) restored cognitive and behavioral impairments in aged microcebes, an animal model that mimics AD pathology. Except M1 agonists, there are no reports on compounds having combined effects, e.g., amelioration of cognition dysfunction and beneficial modulation of APPs together with tau phosphorylation. This unique property of M1 agonists to alter different aspects associated with AD pathogenesis could represent the most remarkable, yet unexplored, or even ignored, clinical value of such drugs. Finally, EVOXAC™ (Cevimeline, AF102B) was recently approved by the FDA for the treatment of dry mouth in Sjogren syndrome, an autoimmune disease that affects exocrine glands. Drug Dev. Res. 50:291–297, 2000. © 2000 Wiley-Liss, Inc.

Therapeutic Strategies in Alzheimer’s Disease: Ml Muscarinic Agonists

The cholinergic hypofunction in Alzheimer's disease (AD) appears to be linked with two other major hallmarks of this disease, beta-amyloid and hyperphosphorylated tau protein. Formation of beta-amyloids might impair the coupling of M1 muscarinic acetylcholine receptors (mAChR) with G-proteins. This can lead to decreased signal transduction, a decrease of trophic and non-amyloidogenic amyloid precursor protein (APPs) and generation of more beta-amyloids, aggravating further the cholinergic deficiency. This review is an attempt to explore the M1 mAChR regulation of beta-amyloid metabolism, tau hyperphosphorylation and cognitive functions. The therapeutic potential of M1-selective muscarinic agonists including AF102B, AF150(S), AF267B (the AF series) is evaluated and compared, when possible, with several FDA-approved acetylcholinesterase inhibitors. These M1 agonists can elevate APPs, decrease tau protein phosphorylation/hyperphosphorylation in vitro and in vivo and restore cognitive impairments in several animal models for AD. Except for the M1 agonists, no other compounds were reported yet with combined effects; e.g., amelioration of cognition dysfunction and beneficial modulation of APPs/beta-amyloid together with tau hyperphosphorylation/phosphorylation. This property of M1 agonists to alter different aspects associated with AD pathogenesis could represent the most remarkable clinical value of such drugs.

Corticotropin-Releasing Hormone-Mediated Neuroprotection against Oxidative Stress Is Associated with the Increased Release of Non-amyloidogenic Amyloid Precursor Protein and with the Suppression of Nuclear Factor- B

Corticotropin-Releasing Hormone-Mediated Neuroprotection against Oxidative Stress Is Associated with the Increased Release of Non-amyloidogenic Amyloid Precursor Protein and with the Suppression of Nuclear Factor- B

Cognitive changes and modified processing of amyloid precursor protein in the cortical and hippocampal system after cholinergic synapse loss and muscarinic receptor activation.

A number of in vitro studies have shown that activation of muscarinic receptors by cholinergic agonists stimulates the nonamyloidogenic, alpha-secretase-processing pathway of amyloid precursor protein (APP). To determine whether increased cholinergic neurotransmission can modify the APP processing in vivo, we administered a muscarinic receptor agonist (RS86) to normal or aged rats and rats with severe basal forebrain cholinergic deficits (induced by 192 IgG-saporin). The levels of the cell-associated APP in neocortex, hippocampus, and striatum, as well as the secreted form of APP (APPs) in cerebrospinal fluid, were examined by Western blots. Additionally, we investigated the association between the altered APP levels and behavioral deficits caused by cholinergic lesions. We found that treatment with muscarinic receptor agonist resulted in decreased APP levels in neocortex and hippocampus and increased levels of APPs in cerebrospinal fluid. Regulation of APP processing by the muscarinic agonist treatment occurred not only in normal rats, but also in aged and cholinergic denervated rats that model this aspect of Alzheimer's disease. Interestingly, we found that elevation of APP in neocortex correlated with the cognitive deficits in water-maze testing of rats with cholinergic dysfunction. These data indicate that increased cholinergic neurotransmission can enhance nonamyloidogenic APP processing in intact and lesioned rats and that APP may be involved in cognitive performance.

Intracellular and cell-surface distribution of amyloid precursor protein in cortical astrocytes

Amyloid peptides that aggregate to form plaques in Alzheimer’s disease are derived from secretory processing of the amyloid precursor protein (APP). Transport of APP to the cell surface may be prerequisite for non-amyloidogenic APP processing and the secretion of soluble APP (APPs), while missorting or reinternalization of APP to intracellular compartments can promote amyloid formation. In cultured astrocytes, APP mRNA and holoprotein are increased by elevations in cAMP levels, and 8-Bromo-cAMP promotes process formation on these cells. We now report that treatment of cultured astrocytes with 8-Bromo-cAMP increased intracellular and cell surface APP in the soma and perinuclear region as detected by immunolabeling with monoclonal antibody 22C11 and polyclonal antibody Kunitz-type protease inhibitor (KPI) (against the N-terminus and KPI domain of APP, respectively) and led to intense but discontinuous labelling of APP on the surface of astrocytic processes. Northern and Western blot analyses confirmed that 8-Bromo-cAMP treatment of cultured astrocytes also increased APP mRNA and KPI-containing APP holoprotein, implying that the intense APP immunolabeling observed in 8-Bromo-cAMP treated astrocytes was not derived from truncated forms of APP (e.g., APPs), but reflected high levels of APP holoprotein containing intact amyloid peptides. Discontinuous cell surface staining in process-bearing astrocytes may be caused by miscompartmentalization of APP related to rearrangement of the cytoskeleton. Inasmuch as intracellular APP is not accessible for non-amyloidogenic processing, we suggest that the increased immunoreactivity of intracellular APP in process-bearing astrocytes may predispose the cells to increased amyloid production.

Benzolactam (BL) enhances sAPP secretion in fibroblasts and in PC12 cells.

Activation of protein kinase C is known to favor the alpha-secretase processing of the Alzheimer's disease (AD) amyloid precursor protein (APP), resulting in the generation of non-amyloidogenic soluble APP (sAPP). Consequently, the relative secretion of amyloidogenic Abeta1-40 and Abeta1-42(3) is reduced. This is particularly relevant since fibroblasts and other cells expressing APP and presenilin AD mutations secrete increased amounts of total Abeta and/or increased ratios of Abeta1-42(3)/Abeta1-40. Interestingly, PKC defects have been found in AD brain alpha and beta isoforms) and in fibroblasts (alpha isoform) from AD patients. Here, we use a novel PKC activator (benzolactam, BL) with improved selectivity for the alpha, beta and gamma isoforms to enhance sAPP secretion in fibroblasts from AD patients and in PC12 cells. Incubation (2 h) of AD fibroblasts with BL (1 and 10 microM) resulted in significant increases of sAPP secretion over basal levels. sAPP secretion in BL-treated AD cells was also slightly higher compared to control BL-treated fibroblasts, which only showed significant increases of sAPP secretion after treatment with 10 microM BL. Staurosporine (a PKC inhibitor) eliminated the effects of BL in both control and AD fibroblasts. BL and a related compound (LQ12) also caused an approximately 3-fold sAPP secretion in PC12 cells. The use of a novel and possibly non-tumorigenic PKC activator may prove useful to favor non-amyloidogenic APP processing and is, therefore, of potential therapeutic value.

The up-regulation of metabotropic glutamate receptor 5 (mGluR5) in Down's syndrome brains.

We investigated the expression of metabotropic glutamate receptor 5 (mGluR5), a subtype of group I mGluRs, in the cerebral cortex of cases with Down's syndrome (DS), using immunohistochemistry and immunoblotting with this receptor. The up-regulation of mGluR5 was observed in DS by immunohistochemistry, and atrophic pyramidal neurons were immunolabelled in elderly DS cases. Western blotting confirmed the increased expression in DS brains. Since group I mGluR regulates the metabolism of amyloid precursor protein (APP) and accelerates its processing into non-amyloidogenic APP, the overexpression of mGluR5 may be related to the pathological state of APP metabolism in DS.