RESUMO
The NAD(+)-dependent protein deacetylase SIRT1 is linked to cellular survival pathways by virtue of keeping the tumor suppressor gene p53 and members of the forkhead transcription factor family deacetylated. To validate SIRT1 as a therapeutic anti-cancer target, we performed immunohistochemistry experiments to study the in vivo expression of SIRT1 in cancer specimens. We show that human SIRT1 is highly expressed in cancer cell lines as well as in tissue samples from colon carcinoma patients. Interestingly, there is a strong cytosolic component in the SIRT1 expression pattern. We further characterized SIRT1 in p53-wild-type and -mutant cell lines and show that SIRT1 mRNA-knockdown leads to a p53-independent decrease of cell proliferation and induction of apoptosis. In addition, SIRT1 expression has been found to be inducible upon DNA damage. A previously discovered small molecule SIRT1 inhibitor with nanomolar in vitro activity has been tested in cancer relevant assays. The SIRT1 inhibitory compound showed no potent anti-proliferative activity despite hitting its molecular target within tumor cells. From these studies we conclude that it may not be sufficient to block the catalytic function of SIRT1, and that its survival effects may be mainly brought about by means other then the deacetylase function. The increased cytosolic expression of SIRT1 in cancer cells could be an indicator of such novel functions.
Assuntos
Neoplasias/metabolismo , Sirtuínas/metabolismo , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citosol/metabolismo , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Mutação , Neoplasias/genética , Neoplasias/patologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirtuína 1 , Sirtuínas/genética , Análise Serial de Tecidos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase SIRT1 has been linked to fatty acid metabolism via suppression of peroxysome proliferator-activated receptor gamma (PPAR-gamma) and to inflammatory processes by deacetylating the transcription factor NF-kappaB. First, modulation of SIRT1 activity affects lipid accumulation in adipocytes, which has an impact on the etiology of a variety of human metabolic diseases such as obesity and insulin-resistant diabetes. Second, activation of SIRT1 suppresses inflammation via regulation of cytokine expression. Using high-throughput screening, the authors identified compounds with SIRT1 activating and inhibiting potential. The biological activity of these SIRT1-modulating compounds was confirmed in cell-based assays using mouse adipocytes, as well as human THP-1 monocytes. SIRT1 activators were found to be potent lipolytic agents, reducing the overall lipid content of fully differentiated NIH L1 adipocytes. In addition, the same compounds have anti-inflammatory properties, as became evident by the reduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In contrast, a SIRT1 inhibitory compound showed a stimulatory activity on the differentiation of adipocytes, a feature often linked to insulin sensitization.
Assuntos
Anti-Inflamatórios/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Quinoxalinas/química , Sirtuínas/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Insulina , Lipogênese/efeitos dos fármacos , Camundongos , Estrutura Molecular , Sirtuína 1 , Sirtuínas/agonistas , Sirtuínas/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
It has been demonstrated that GSK3beta is involved in Alzheimer Disease (AD) pathogenesis. In order to understand the underlying mechanism, we have generated and characterized transgenic mice in which the constitutively active human GSK3beta (with S9A mutation) was overexpressed in the brain under the control of the platelet-derived growth factor (PDGF) B-chain promoter. Varying levels of human GSK3betaS9A transgene protein expression was observed in six of the seven founders generated. Line 3083, 3107, 3112 and 3125 displayed higher GSK3betaS9A protein expression levels. Immunostaining analysis demonstrated that transgene expression was observed mainly in cortex and hippocampus of transgenic brain. Expression of human GSK3beta transgene did not significantly change the brain total GSK3beta protein levels in any of the generated mouse lines, as comparing to age matched wild type mice. Although significant kinase activity was detected in human GSK3betaS9A transgene protein extracted from brains of all six expressing lines, significant increase in total GSK3betaS9A kinase activity was observed only in the offspring of line 3083 and 3107. By analyzing the offspring from several transgenic mouse lines, including lines other than 3083 and 3107, it was found that overexpressed constitutively active human GSK3betaS9A resulted in hyperphosphorylation of tau and morphology reminiscent of pretangle-like neurons in cortex and hippocampus.
Assuntos
Encéfalo/patologia , Quinase 3 da Glicogênio Sintase/genética , Neurônios/patologia , Proteínas tau/metabolismo , Substituição de Aminoácidos , Animais , Becaplermina , Encéfalo/enzimologia , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/química , Glicogênio Sintase Quinase 3 beta , Hipocampo/enzimologia , Hipocampo/patologia , Humanos , Camundongos , Camundongos Transgênicos , Emaranhados Neurofibrilares/patologia , Neurônios/enzimologia , Fosforilação , Fator de Crescimento Derivado de Plaquetas/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-sis , Proteínas tau/genéticaRESUMO
Lithium is one of the most widely used mood-stabilizing agents for the treatment of bipolar disorder. Although the underlying mechanism(s) of this mood stabilizer remains controversial, recent evidence linking lithium to neurotrophic/neuroprotective effects (Choi and Sung (2000) 1475, 225-230; Davies et al. (2000) 351, 95-105) suggests novel benefits of this drug in addition to mood stabilization. Here, we report that both lithium as well as valproic acid (VPA) inhibit beta-amyloid peptide (Abeta) production in HEK293 cells stably transfected with Swedish amyloid precursor protein (APP)(751) and in the brains of the PDAPP (APP(V717F)) Alzheimer's disease transgenic mouse model at clinically relevant plasma concentrations. Both lithium and VPA are known to be glycogen synthase kinase-3 (GSK3) inhibitors. Our studies reveal that GSK3beta is a potential downstream kinase, which modulates APP processing because inhibition of GSK3 activity by either a dominant negative GSK3beta kinase-deficient construct or GSK3beta antisense oligonucleotide mimics lithium and VPA effects. Moreover, lithium treatment abolished GSK3beta-mediated Abeta increase in the brains of GSK3beta transgenics and reduced plaque burden in the brains of the PDAPP (APP(V717F)) transgenic mice.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Lítio/farmacologia , Processamento de Proteína Pós-Traducional , Doença de Alzheimer/tratamento farmacológico , Animais , Transtorno Bipolar/tratamento farmacológico , Encéfalo/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Transgênicos , Oligonucleotídeos Antissenso/farmacologia , Ácido Valproico/farmacologiaRESUMO
Neurotensin (NT) regulates a variety of biological processes primarily through interaction with neurotensin receptor-1 (NTR1), a heterotrimeric G-protein-coupled receptor (GPCR). Stimulation of NTR1 has been linked to activation of multiple signaling transduction pathways via specific coupling to G(q), G(i/o), or G(s), in various cell systems. However, the function of NT/NTR1 in the regulation of the Akt pathway remains unknown. Here, we report that activation of NTR1 by NT inhibits Akt activity as determined by the dephosphorylation of Akt at both Ser473 and Thr308 in AV12 cells constitutively expressing human NTR1 (NTR1/AV12). The inactivation of Akt by NT was rapid and dose-dependent. This effect of NT was completely blocked by the specific NTR1 antagonist, (S)-(+)-[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)-carbonylamino] cyclohexylacetic acid (SR 48527), but unaffected by the less active enantiomer ((R)-(-)-[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)-carbonylamino] cyclohexylacetic acid (SR 49711)), indicating the stereospecificity of NTR1 in the negative regulation of Akt. In addition, NT prevented insulin- and epidermal growth factor (EGF)-mediated Akt activation. Our results provide insight into the role of NT in the modulation of Akt signaling and the potential physiological significance of Akt regulation by NT.
Assuntos
Neurotensina/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Animais , Linhagem Celular Tumoral , Toxina da Cólera/farmacologia , Cricetinae , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/farmacologia , Humanos , Insulina/farmacologia , Antagonistas da Insulina/química , Antagonistas da Insulina/farmacologia , Isoquinolinas/farmacologia , Neurotensina/química , Toxina Pertussis/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores de Neurotensina/química , Sulfonamidas/farmacologia , TransfecçãoRESUMO
Although Alzheimer's disease pathologically affects the brain, familial Alzheimer's disease associated mutations of beta-amyloid precursor protein and presenilin are ubiquitously expressed and therefore aberrant intracellular signals, separate from but similar to, the brain may be expected. Here, we report selective down regulation of the serine/threonine kinase, Akt/PKB, concurrent with elevated endogenous GSK3beta kinase activity in familial Alzheimer's disease beta-amyloid precursor protein expressing human embryonic kidney (HEK) and familial Alzheimer's disease presenilin lymphoblast cells. Further, familial Alzheimer's disease presenilin in the human lymphoblast was associated with beta-catenin destabilization. Moreover, limited immunohistochemistry analysis reveals Akt/PKB in a subset of neurofibrillary tangles where GSK3beta and tau have been reported to co-localize, suggesting a possible Akt/GSK3beta and tau interaction in vivo. Our data suggest that familial Alzheimer's disease mutants of beta-amyloid precursor protein and presenilin signal, at least in part, through the Akt/GSKbeta pathway and that Akt/GSK3beta-mediated signalling may contribute to the underlying Alzheimer's disease pathogenesis induced by familial Alzheimer's disease mutants.
Assuntos
Doença de Alzheimer/enzimologia , Precursor de Proteína beta-Amiloide/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Idoso , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Feminino , Glicogênio Sintase Quinase 3 beta , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Imuno-Histoquímica , Rim/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Fosforilação , Presenilina-1 , Proteínas Proto-Oncogênicas c-akt , Transativadores/metabolismo , beta Catenina , Proteínas tau/metabolismoRESUMO
Glycogen synthase kinase-3 (GSK3) and cyclin-dependent kinase 5 (CDK5) are related serine/threonine kinases that have been well studied for their role in tau hyperphosphorylation, however, little is known about their significance in APP processing. Here we report that GSK3 and CDK5 are involved in APP processing in a divergent manner. Specific inhibition of cellular GSK3 by lithium or GSK3beta antisense elicits a reduction in Abeta. Conversely, negative modulation of cellular CDK5 activity by CDK5 inhibitor, roscovitine, or CDK5 antisense stimulates Abeta production. Neither GSK3 nor CDK5 inhibition by these means significantly affected cellular APP levels or APP maturation. Moreover, oral administration of lithium significantly reduces Abeta production whereas direct ICV administration of roscovitine augmented Abeta production in the brains of PDAPP (APP(V717F)) mice. Our data support a function for both GSK3 and CDK5 in APP processing, further implicating these two kinases in the pathogenesis of Alzheimer's disease.
Assuntos
Doença de Alzheimer/enzimologia , Precursor de Proteína beta-Amiloide/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Animais , Linhagem Celular , Quinase 5 Dependente de Ciclina , Relação Dose-Resposta a Droga , Lítio/farmacologia , Camundongos , Camundongos Transgênicos , Purinas/farmacologia , RoscovitinaRESUMO
A subset of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to preferentially reduce the secretion of the highly amyloidogenic, 42-residue amyloid-beta peptide Abeta42. We found that Rho and its effector, Rho-associated kinase, preferentially regulated the amount of Abeta42 produced in vitro and that only those NSAIDs effective as Rho inhibitors lowered Abeta42. Administration of Y-27632, a selective Rock inhibitor, also preferentially lowered brain levels of Abeta42 in a transgenic mouse model of Alzheimer's disease. Thus, the Rho-Rock pathway may regulate amyloid precursor protein processing, and a subset of NSAIDs can reduce Abeta42 through inhibition of Rho activity.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Fragmentos de Peptídeos/metabolismo , Sulindaco/análogos & derivados , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Amidas/farmacologia , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Guanosina Trifosfato/metabolismo , Humanos , Ibuprofeno/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Fosfatos de Poli-Isoprenil/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Sesquiterpenos , Transdução de Sinais , Sulindaco/farmacologia , Transfecção , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) resulting in the inhibition of GSK-3 kinase activity and increase of beta-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cell-matrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Células PC12 , Proteínas/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas de Repetições Ricas em Leucina , Dados de Sequência Molecular , Fosforilação , Proteínas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Distribuição Tecidual , Proteínas Wnt , Dedos de Zinco , beta CateninaRESUMO
Recently, LiCl has been shown to inhibit amyloid beta peptide secretion in association with diminished glycogen synthase kinase beta (GSK3beta) activity. However, it remains unclear if direct inhibition of GSK3beta activity will result in decreased Abeta production. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) protein is a negative regulator of GSK3alpha/beta kinase activity. To examine whether direct inhibition of GSK3alpha/beta kinase activity can lower Abeta production, a FRAT1 peptide was expressed in swAPP(751) cells that produce high levels of Abeta. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP(751) cells increases both GSK3alpha and beta phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total Abeta and Abeta(1-42) was significantly decreased. Thus, we provide evidence that direct regulation of GSK3alpha/beta by FRAT1 peptide significantly decreases Abeta production in swAPP(751) cells.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Proteínas de Transporte , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Peptídeos beta-Amiloides/genética , Western Blotting , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Vetores Genéticos/genética , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Serina/metabolismo , TransfecçãoRESUMO
The increased intracellular levels and aberrant processing of the amyloid precursor protein (APP) are associated with beta-amyloid peptide (A beta) production, cerebrovascular amyloid deposition, and amyloid plaque formation. Here we report that APP level, soluble APP (sAPP) secretion, and A beta production in HEK293 cells transfected with either wild-type APP(751) or APP(751) carrying the Swedish mutation are all elevated by hepatocyte growth factor (HGF). We investigated the potential molecular mechanisms underlying the HGF effect. Our data show that HGF stimulated extended activation of extracellular signal-regulated protein kinases (ERK1/2). Pretreatment of cells with inhibitors (UO126 or PD98059) for MEK, the upstream kinase of ERK1/2, abolished ERK1/2 activation evoked by HGF, and abrogated HGF-induced increases in APP levels and sAPP secretion. In addition, transient expression of active MEK1 activated ERK1/2 and increased intracellular APP levels and sAPP secretion. Inhibition of ERK1/2 activity, however, failed to block HGF-stimulated A beta production. Consistently, transient expression of active MEK1 did not increase A beta accumulation. Taken together, these results suggest that: (1) HGF regulates the intracellular levels of APP and the secretion of sAPP and A beta; (2) the modulation of APP levels and sAPP secretion induced by HGF is mediated via the MEK1/ERK1/2 signaling pathway; (3) HGF-stimulated A beta production is independent of ERK activity and, therefore, independent of HGF-evoked elevation of intracellular APP levels.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/genética , Butadienos/farmacologia , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Nitrilas/farmacologia , Transdução de Sinais , TransfecçãoRESUMO
The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP(beta) (sAPP(beta)) and beta-amyloid peptide (Abeta), the major component of senile plaques in patient brains inflicted with Alzheimer's disease (AD), or soluble APP(alpha) (sAPP(alpha)) and a peptide smaller than Abeta. One of the protein kinases known to phosphorylate APP(Thr668) is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative effects of p35 and p25 on APP(Thr668) phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP(Thr668) phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP(Thr668) phosphorylation, specifically, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these differential effects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of Abeta, sAPP(beta), as well as sAPP(alpha). These results confirm the involvement of Cdk5 in APP processing, and suggest that p35- and p25-mediated Cdk5 activities lead to discrete APP phosphorylation.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linhagem Celular , Vetores Genéticos , Humanos , Rim , Neuroblastoma , Fosforilação , Fosfotreonina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Treonina , Transfecção , Células Tumorais CultivadasRESUMO
Alzheimer's disease is characterized pathologically by extracellular amyloid beta protein (Abeta) deposition in the brain. The Abeta peptide, a 39-42 amino acid fragment, is derived from defined proteolysis of the amyloid precursor protein (APP) [Glenner et al., Appl. Pathol. 2 (1984) 357-369; Selkoe, Neuron 6 (1991) 487-498] and is the primary component of senile plaques. Although it is known that intracellular APP is subjected to posttranslational modification, the molecular mechanism that regulates the APP processing is not completely clear. In the present study, we demonstrates that H89, a specific inhibitor for cAMP dependent protein kinase A (PKA), inhibits Abeta production and APP secretion in a dose dependent manner in cells stably transfected with human APP bearing a 'Swedish mutation'. Concurrent with the effect, H89 inhibits C-terminal fragment of the APP. We also found that the PKA inhibitor abolishes the mature form of intracellular APP and accumulates the immature form. Finally, direct administration of H89 into brains of transgenic mice overexpressing human APP shows that the compound inhibits Abeta production in the hippocampal region. Our data suggests that PKA plays an important role in the maturation of APP associated with APP processing.