RESUMO
Abnormal calcium signaling is a central pathological component of Alzheimer's disease (AD). Here, we describe the identification of a class of compounds called ReS19-T, which are able to restore calcium homeostasis in cell-based models of tau pathology. Aberrant tau accumulation leads to uncontrolled activation of store-operated calcium channels (SOCCs) by remodeling septin filaments at the cell cortex. Binding of ReS19-T to septins restores filament assembly in the disease state and restrains calcium entry through SOCCs. In amyloid-ß and tau-driven mouse models of disease, ReS19-T agents restored synaptic plasticity, normalized brain network activity, and attenuated the development of both amyloid-ß and tau pathology. Our findings identify the septin cytoskeleton as a potential therapeutic target for the development of disease-modifying AD treatments.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Cálcio , Homeostase , Fármacos Neuroprotetores , Septinas , Proteínas tau , Animais , Humanos , Camundongos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/efeitos dos fármacos , Modelos Animais de Doenças , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Septinas/metabolismo , Proteínas tau/metabolismoRESUMO
BACKGROUND: Neuronal Ca2+ dyshomeostasis and hyperactivity play a central role in Alzheimer's disease pathology and progression. Amyloid-beta together with non-genetic risk-factors of Alzheimer's disease contributes to increased Ca2+ influx and aberrant neuronal activity, which accelerates neurodegeneration in a feed-forward fashion. As such, identifying new targets and drugs to modulate excessive Ca2+ signalling and neuronal hyperactivity, without overly suppressing them, has promising therapeutic potential. METHODS: Here we show, using biochemical, electrophysiological, imaging, and behavioural tools, that pharmacological modulation of Rap1 signalling by inhibiting its interaction with Pde6δ normalises disease associated Ca2+ aberrations and neuronal activity, conferring neuroprotection in models of Alzheimer's disease. RESULTS: The newly identified inhibitors of the Rap1-Pde6δ interaction counteract AD phenotypes, by reconfiguring Rap1 signalling underlying synaptic efficacy, Ca2+ influx, and neuronal repolarisation, without adverse effects in-cellulo or in-vivo. Thus, modulation of Rap1 by Pde6δ accommodates key mechanisms underlying neuronal activity, and therefore represents a promising new drug target for early or late intervention in neurodegenerative disorders. CONCLUSION: Targeting the Pde6δ-Rap1 interaction has promising therapeutic potential for disorders characterised by neuronal hyperactivity, such as Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Neuroproteção/fisiologia , Fármacos Neuroprotetores/uso terapêutico , Complexo Shelterina , Transdução de Sinais/fisiologiaRESUMO
We have developed a yeast-based model recapitulating neurotoxicity of alpha-synuclein fibrilization. This model recognized metal ions, known risk factors of alpha-synucleinopathy, as stimulators of alpha-synuclein aggregation and cytotoxicity. Elimination of Yca1 caspase activity augmented both cytotoxicity and inclusion body formation, suggesting the involvement of apoptotic pathway components in toxic alpha-synuclein amyloidogenesis. Deletion of hydrophobic amino acids at positions 66-74 in alpha-synuclein reduced its cytotoxicity but, remarkably, did not lower the levels of insoluble alpha-synuclein, indicating that noxious alpha-synuclein species are different from insoluble aggregates. A compound screen aimed at finding molecules with therapeutic potential identified flavonoids with strong activity to restrain alpha-synuclein toxicity. Subsequent structure-activity analysis elucidated that these acted by virtue of anti-oxidant and metal-chelating activities. In conclusion, this yeast-cell model as presented allows not only fundamental studies related to mechanisms of alpha-synuclein-instigated cellular degeneration, but is also a valid high-throughput identification tool for novel neuroprotective agents.
Assuntos
Encefalopatias/tratamento farmacológico , Encefalopatias/patologia , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/metabolismo , Encefalopatias/metabolismo , Inibidores de Caspase , Caspases/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/metabolismo , Catequina/uso terapêutico , Flavonoides/química , Flavonoides/metabolismo , Flavonoides/uso terapêutico , Humanos , Metais/metabolismo , Estrutura Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Risco , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadeRESUMO
A yeast model was generated to study the mechanisms and phenotypical repercussions of expression of alpha-synuclein as well as the coexpression of protein tau. The data show that aggregation of alpha-synuclein is a nucleation-elongation process initiated at the plasma membrane. Aggregation is consistently enhanced by dimethyl sulfoxide, which is known to increase the level of phospholipids and membranes in yeast cells. Aggregation of alpha-synuclein was also triggered by treatment of the yeast cells with ferrous ions, which are known to increase oxidative stress. In addition, data are presented in support of the hypothesis that degradation of alpha-synuclein occurs via autophagy and proteasomes and that aggregation of alpha-synuclein disturbs endocytosis. Reminiscent of observations in double-transgenic mice, coexpression of alpha-synuclein and protein tau in yeast cells is synergistically toxic, as exemplified by inhibition of proliferation. Taken together, the data show that these yeast models recapitulate major aspects of alpha-synuclein aggregation and cytotoxicity, and offer great potential for defining the underlying mechanisms of toxicity and synergistic actions of alpha-synuclein and protein tau.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/fisiologia , Dimetil Sulfóxido/farmacologia , Endocitose/fisiologia , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/química , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sinucleínas , alfa-Sinucleína , Proteínas tau/metabolismoRESUMO
Active vaccination with amyloid peptides shows promise for the treatment and prevention of Alzheimer's disease (AD). Several studies in transgenic mouse models of AD have revealed the potency of vaccination to prevent or even clear amyloid plaques from mouse brain. However, the idea that soluble oligomeric species of beta-amyloid (Abeta), rather than plaques, trigger the disease has gained momentum, and current active vaccination strategies affect the levels of total or soluble brain Abeta little or not at all. We describe an active vaccination method based on Abeta1-16 presented on the surface of virosomes, which triggered a dramatic decrease in both soluble Abeta40 (75% reduction; p=0.01) and soluble Abeta42 (62% reduction; p=0.03) in a double transgenic mouse model of AD. Whereas Abeta40 and Abeta42 levels in the insoluble fraction tended to be reduced (by 30% and 27%, respectively), the number of thioflavine-S-positive amyloid plaques was not affected. The high specific antibody responses, obtained without eliciting T-cell reactivity, demonstrate that immunostimulating reconstituted influenza virosomes are a promising antigen carrier system against the neuropathology of AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/imunologia , Placa Amiloide/imunologia , Vacinas Virossomais/imunologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Animais , Biomarcadores , Química Encefálica , Modelos Animais de Doenças , Imunização , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Placa Amiloide/patologia , Distribuição AleatóriaRESUMO
The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.
Assuntos
Glucose/farmacologia , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Domínio Catalítico , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
A novel series of 2-aminothiazoles with strong protection in an Alzheimer's disease (AD) model comprising tau-induced neuronal toxicity is disclosed. These derivatives can be synthesized in one-pot and a small SAR of the substitution within these series afforded several compounds that counteracted tau-induced cell toxicity at nanomolar concentrations. These congeners therefore have strong potential as possible treatment for Alzheimer's disease and other related tauopathies.
Assuntos
Doença de Alzheimer/patologia , Modelos Biológicos , Neurônios/efeitos dos fármacos , Tiazóis/farmacologia , Proteínas tau/fisiologia , Linhagem Celular , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Neurônios/patologia , Tiazóis/químicaRESUMO
Apolipoprotein E4 (ApoE4) is associated with Alzheimer's disease by unknown mechanisms. We generated six transgenic mice strains expressing human ApoE4 in combination with mutant amyloid precursor protein (APP) and mutant presenilin-1 (PS1) in single-, double-, or triple-transgenic combinations. Diffuse, but not dense, amyloid plaque-load in subiculum and cortex was increased by neuronal but not glial ApoE4 in old (15 months) double-transgenic mice, whereas both diffuse and dense plaques formed in thalamus in both genotypes. Neuronal and glial ApoE4 promoted cerebral amyloid angiopathy as extensively as mutant PS1 but with pronounced regional differences: cortical angiopathy was induced by neuronal ApoE4 while thalamic angiopathy was again independent of ApoE4 source. Angiopathy correlated more strongly with soluble Abeta40 and Abeta42 levels in cortex than in thalamus throughout the six genotypes. Neither neuronal nor glial ApoE4 affected APP proteolytic processing, as opposed to mutant PS1. Neuronal ApoE4 increased soluble amyloid levels more than glial ApoE4, but the Abeta42/40 ratios were similar, although significantly higher than in single APP transgenic mice. We conclude that although the cellular origin of ApoE4 differentially affects regional amyloid pathology, ApoE4 acts on the disposition of amyloid peptides downstream from their excision from APP but without induction of tauopathy.
Assuntos
Apolipoproteínas E/biossíntese , Encéfalo/patologia , Angiopatia Amiloide Cerebral/genética , Neuroglia/metabolismo , Neurônios/metabolismo , Placa Amiloide/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apolipoproteína E4 , Western Blotting , Encéfalo/irrigação sanguínea , Química Encefálica , Angiopatia Amiloide Cerebral/patologia , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mutação , Placa Amiloide/metabolismo , Presenilina-1 , Proteínas tau/metabolismoRESUMO
BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that primarily strikes the elderly. Studies in both humans and animal models have linked the consumption of cholesterol and saturated fats with amyloid-beta (Abeta) deposition and development of AD. Yet, these studies did not examine high fat diets in combination with reduced carbohydrate intake. Here we tested the effect of a high saturated fat/low carbohydrate diet on a transgenic mouse model of AD. RESULTS: Starting at three months of age, two groups of female transgenic mice carrying the "London" APP mutation (APP/V717I) were fed either, a standard diet (SD) composed of high carbohydrate/low fat chow, or a ketogenic diet (KD) composed of very low carbohydrate/high saturated fat chow for 43 days. Animals fed the KD exhibited greatly elevated serum ketone body levels, as measured by beta-hydroxybutyrate (3.85 +/- 2.6 mM), compared to SD fed animals (0.29 +/- 0.06 mM). In addition, animals fed the KD lost body weight (SD 22.2 +/- 0.6 g vs. KD 17.5 +/- 1.4 g, p = 0.0067). In contrast to earlier studies, the brief KD feeding regime significantly reduced total brain Abeta levels by approximately 25%. Despite changes in ketone levels, body weight, and Abeta levels, the KD diet did not alter behavioral measures. CONCLUSION: Previous studies have suggested that diets rich in cholesterol and saturated fats increased the deposition of Abeta and the risk of developing AD. Here we demonstrate that a diet rich in saturated fats and low in carbohydrates can actually reduce levels of Abeta. Therefore, dietary strategies aimed at reducing Abeta levels should take into account interactions of dietary components and the metabolic outcomes, in particular, levels of carbohydrates, total calories, and presence of ketone bodies should be considered.
RESUMO
Hyperphosphorylation and aggregation of protein tau are typical for neurodegenerative tauopathies, including Alzheimer's disease (AD). We demonstrate here that human tau expressed in yeast acquired pathological phosphoepitopes, assumed a pathological conformation, and formed aggregates. These processes were modulated by yeast kinases Mds1 and Pho85, orthologues of GSK-3beta and cdk5, respectively. Surprisingly, inactivation of Pho85 increased phosphorylation of tau-4R, concomitant with increased conformational change defined by antibody MC1 and a 40-fold increase in aggregation. Soluble protein tau, purified from yeast lacking PHO85, spontaneously and rapidly formed tau filaments in vitro. Further fractionation of tau by anion-exchange chromatography yielded a hyperphosphorylated monomeric subfraction, termed hP-tau/MC1, with slow electrophoretic mobility and enriched with all major epitopes, including MC1. Isolated hP-tau/MC1 vastly accelerated in vitro aggregation of wild-type tau-4R, demonstrating its functional capacity to initiate aggregation, as well as its structural stability. Combined, this novel yeast model recapitulates hyperphosphorylation, conformation, and aggregation of protein tau, provides insight in molecular changes crucial in tauopathies, offers a source for isolation of modified protein tau, and has potential for identification of modulating compounds and genes.
Assuntos
Proteínas tau/química , Proteínas tau/metabolismo , Anticorpos , Humanos , Fosforilação , Conformação Proteica , Proteínas Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas tau/genéticaRESUMO
Early studies identified two bona fide protein phosphatase 2A (PP2A)-encoding genes in Saccharomyces cerevisiae, designated PPH21 and PPH22. In addition, three PP2A-related phosphatases, encoded by PPH3, SIT4 and PPG1, have been identified. All share as much as 86% sequence similarity at the amino acid level. This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4 regulation will also be discussed. Whereas a role for PP2A in yeast morphology and cell cycle has been readily recognized, uncovering its function in yeast signal transduction is a more recent breakthrough. Via their interaction with phosphorylated Tap42, PP2A and Sit4 play a pivotal role in target of rapamycin (TOR) signalling. PPH22 overexpression mimics overactive cAMP-PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets. The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling. We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient-induced signalling in baker's yeast.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Meios de Cultura , Proteínas Fúngicas/metabolismo , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2 , Saccharomyces cerevisiae/genética , Sirolimo/metabolismoRESUMO
The Saccharomyces cerevisiae type 2A protein phosphatase (PP2A) Pph22p differs from the catalytic subunits of PP2A (PP2Ac) present in mammals, plants and Schizosaccharomyces pombe by a unique N-terminal extension of approximately 70 amino acids. We have overexpressed S. cerevisiae Pph22p and its N-terminal deletion mutant Delta N-Pph22p in the GS115 strain of Pichia pastoris and purified these enzymes to apparent homogeneity. Similar to other heterologous systems used to overexpress PP2Ac, a low yield of an active enzyme was obtained. The recombinant enzymes designed with an 8 x His-tag at their N-terminus were purified by ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on Ni2+-nitrilotriacetic acid agarose. Comparison of biochemical properties of purified Pph22p and Delta N-Pph22p with purified human 8 x His PP2Ac identified similarities and differences between these two enzymes. Both enzymes displayed similar specific activities with 32P-labelled phosphorylase a as substrate. Furthermore, selected inhibitors and metal ions affected their activities to the same extend. In contrast to the mammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not Delta N-Pph22p, interacted strongly with protamine. Also with regard to the effects of protamine and polylysine on phosphatase activity Pph22p, but not Delta N-Pph22p, behaved similarly to the PP2Ac-PR65 dimer, indicating a regulatory role for the N-terminal extension of Pph22p. The N-terminal extension appears also responsible for interactions with phospholipids. Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents. These properties make the S. cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far.
Assuntos
Mamíferos/metabolismo , Fosfoproteínas Fosfatases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Clonagem Molecular , Inibidores Enzimáticos/farmacologia , Humanos , Lipossomos , Mamíferos/genética , Dados de Sequência Molecular , Oxirredução , Fosfolipídeos/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosforilase a/metabolismo , Pichia , Polilisina/farmacologia , Protaminas/farmacologia , Proteína Fosfatase 2 , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP(3)) in yeast is rapidly transformed into IP(4) and IP(5) by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP(5) into IP(6). In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP(3) accumulation upon glucose addition was completely absent in the plc1Delta strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP(2) and generation of IP(3), seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP(3) signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.
Assuntos
Sinalização do Cálcio , Inositol 1,4,5-Trifosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , AMP Cíclico/metabolismo , Genes Fúngicos , Glucose/farmacologia , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Alzheimer's disease (AD) characteristically presents with early memory loss. Regulation of K(+) channels, calcium homeostasis, and protein kinase C (PKC) activation are molecular events that have been implicated during associative memory which are also altered or defective in AD. PKC is also involved in the processing of the amyloid precursor protein (APP), a central element in AD pathophysiology. In previous studies, we demonstrated that benzolactam (BL), a novel PKC activator, reversed K(+) channels defects and enhanced secretion of APP alpha in AD cells. In this study we present data showing that another PKC activator, bryostatin 1, at subnanomolar concentrations dramatically enhances the secretion of the alpha-secretase product sAPP alpha in fibroblasts from AD patients. We also show that BL significantly increased the amount of sAPP alpha and reduced A beta 40 in the brains of APP[V717I] transgenic mice. In a more recently developed AD double-transgenic mouse, bryostatin was effective in reducing both brain A beta 40 and A beta 42. In addition, bryostatin ameliorated the rate of premature death and improved behavioral outcomes. Collectively, these data corroborate PKC and its activation as a potentially important means of ameliorating AD pathophysiology and perhaps cognitive impairment, thus offering a promising target for drug development. Because bryostatin 1 is devoid of tumor-promoting activity and is undergoing numerous clinical studies for cancer treatment in humans, it might be readily tested in patients as a potential therapeutic agent for Alzheimer's disease.