Current and emerging avenues for Alzheimer's disease drug targets.

Alzheimer's disease (AD), the most frequent cause of dementia, is escalating as a global epidemic, and so far, there is neither cure nor treatment to alter its progression. The most important feature of the disease is neuronal death and loss of cognitive functions, caused probably from several pathological processes in the brain. The main neuropathological features of AD are widely described as amyloid beta (Aß) plaques and neurofibrillary tangles of the aggregated protein tau, which contribute to the disease. Nevertheless, AD brains suffer from a variety of alterations in function, such as energy metabolism, inflammation and synaptic activity. The latest decades have seen an explosion of genes and molecules that can be employed as targets aiming to improve brain physiology, which can result in preventive strategies for AD. Moreover, therapeutics using these targets can help AD brains to sustain function during the development of AD pathology. Here, we review broadly recent information for potential targets that can modify AD through diverse pharmacological and nonpharmacological approaches including gene therapy. We propose that AD could be tackled not only using combination therapies including Aß and tau, but also considering insulin and cholesterol metabolism, vascular function, synaptic plasticity, epigenetics, neurovascular junction and blood-brain barrier targets that have been studied recently. We also make a case for the role of gut microbiota in AD. Our hope is to promote the continuing research of diverse targets affecting AD and promote diverse targeting as a near-future strategy.

Can LDL cholesterol be too low? Possible risks of extremely low levels.

Following the continuous accumulation of evidence supporting the beneficial role of reducing low-density lipoprotein cholesterol (LDL-C) levels in the treatment and prevention of atherosclerotic cardiovascular disease and its complications, therapeutic possibilities now exist to lower LDL-C to very low levels, similar to or even lower than those seen in newborns and nonhuman species. In addition to the important task of evaluating potential side effects of such treatments, the question arises whether extremely low LDL-C levels per se may provoke adverse effects in humans. In this review, we summarize information from studies of human cellular and organ physiology, phenotypic characterization of rare genetic diseases of lipid metabolism, and experience from clinical trials. Specifically, we emphasize the importance of the robustness of the regulatory systems that maintain balanced fluxes and levels of cholesterol at both cellular and organismal levels. Even at extremely low LDL-C levels, critical capacities of steroid hormone and bile acid production are preserved, and the presence of a cholesterol blood-brain barrier protects cells in the central nervous system. Apparent relationships sometimes reported between less pronounced low LDL-C levels and disease states such as cancer, depression, infectious disease and others can generally be explained as secondary phenomena. Drug-related side effects including an increased propensity for development of type 2 diabetes occur during statin treatment, whilst further evaluation of more potent LDL-lowering treatments such as PCSK9 inhibitors is needed. Experience from the recently reported and ongoing large event-driven trials are of great interest, and further evaluation including careful analysis of cognitive functions will be important.

Resistance training does not have an effect on cognition or related serum biomarkers in nonagenarians: a randomized controlled trial.

The aim of this randomized controlled trial was to determine the effects of 8-week exercise-intervention on cognition and related serum biochemical markers in nonagenarians. We also studied the effects of a 4-week training cessation ('detraining') period on our study variables. Participants were randomly allocated to a standard-care (control) or intervention (exercise) group [n=20 (16 women)/group]. The intervention focused on supervised, light-to-moderate-intensity aerobic and resistance exercises (mainly leg press), and included 3 weekly sessions. Cognitive status was determined by the mini-mental state examination and geriatric depression scale. We analysed proteins with reported relation with mechanisms behind cognition changes such as serum levels of angiotensin converting enzyme, amyloid-precursor protein, epidermal growth factor, brain-derived neural factor and tumor necrosis factor. No significant change (P>0.05) in any of the variables studied was found following the exercise intervention compared with the standard-care group. Similarly, no significant changes (P>0.05) were observed following the detraining period compared with the standard-care group. Overall changes after the exercise intervention in serum biomarkers were not associated with changes in functional capacity and cognitive measures. An 8-week exercise intervention focusing on resistance exercises neither benefits cognitive function nor affects the levels of the serum proteins analysed in nonagenarians.

Pathways to Alzheimer's disease.

Recent trials of anti-amyloid agents have not produced convincing improvements in clinical outcome in Alzheimer's disease; however, the reason for these poor or inconclusive results remains unclear. Recent genetic data continue to support the amyloid hypothesis of Alzheimer's disease with protective variants being found in the amyloid gene and both common low-risk and rare high-risk variants for disease being discovered in genes that are part of the amyloid response pathways. These data support the view that genetic variability in how the brain responds to amyloid deposition is a potential therapeutic target for the disease, and are consistent with the notion that anti-amyloid therapies should be initiated early in the disease process.

Differences in brain cholesterol metabolism and insulin in two subgroups of patients with different CSF biomarkers but similar white matter lesions suggest different pathogenic mechanisms.

Investigate possible associations of white matter hyperintensities (WMHs) with the metabolism of cholesterol and insulin in two subgroups of patients with memory complaints and different CSF Aß42 and CSF tau levels. 59 patients from the memory clinic at Karolinska Hospital were included. Degree of WMHs was rated using the ARWMC scale and the following biomarkers were measured in CSF and plasma: insulin, cholesterol, lanosterol, lathosterol, and oxidized cholesterol metabolites. The WMHs in CSF control-like group correlated with increased brain cholesterol synthesis and reduced efflux of oxysterols and insulin in CSF. In the CSF AD-like group, the WMHs correlated with increased peripheral cholesterol metabolism. Despite having similar appearance on FLAIR images, the pathogenic mechanisms of WMHS are likely to be different in the two groups investigated.

Ezrin mediates c-Myc actions in prostate cancer cell invasion.

The forced overexpression of c-Myc in mouse prostate and in normal human prostate epithelial cells results in tumor transformation with an invasive phenotype. How c-Myc regulates cell invasion is poorly understood. In this study, we have investigated the interplay of c-Myc and androgens in the regulation of prostate cancer cell invasion. We found that c-Myc induces cell invasion and anchorage-independent growth by regulating ezrin protein expression in the presence of androgens. The activity of the ezrin promoter is controlled by androgens through c-Myc, which binds to a phylogenetically conserved E-Box located in the proximal promoter region. Besides, we also show that ezrin is an important regulator of c-Myc protein levels. These effects are achieved through androgen-induced changes in ezrin phosphorylation, which results in the regulation of downstream signals. These downstream signals involve the modulation of Akt and GSK-3beta activity resulting in increased c-Myc protein synthesis and inhibition of its degradation. In summary, we have shown a key role for ezrin as a mediator of c-Myc-induced tumorigenesis in prostate cancer cells.

Involvement of glutaredoxin-1 and thioredoxin-1 in beta-amyloid toxicity and Alzheimer's disease.

Strong evidence indicates oxidative stress in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Abeta) has been implicated in both oxidative stress mechanisms and in neuronal apoptosis. Glutaredoxin-1 (GRX1) and thioredoxin-1 (TRX1) are antioxidants that can inhibit apoptosis signal-regulating kinase (ASK1). We examined levels of GRX1 and TRX1 in AD brain as well as their effects on Abeta neurotoxicity. We show an increase in GRX1 and a decrease in neuronal TRX1 in AD brains. Using SH-SY5Y cells, we demonstrate that Abeta causes an oxidation of both GRX1 and TRX1, and nuclear export of Daxx, a protein downstream of ASK1. Abeta toxicity was inhibited by insulin-like growth factor-I (IGF-I) and by overexpressing GRX1 or TRX1. Thus, Abeta neurotoxicity might be mediated by oxidation of GRX1 or TRX1 and subsequent activation of the ASK1 cascade. Deregulation of GRX1 and TRX1 antioxidant systems could be important events in AD pathogenesis.

Apolipoprotein E and beta-amyloid (1-42) regulation of glycogen synthase kinase-3beta.

Glycogen synthase kinase-3beta (GSK-3beta) is implicated in regulating apoptosis and tau protein hyperphosphorylation in Alzheimer's disease (AD). We investigated the effects of two key AD molecules, namely apoE (E3 and E4 isoforms) and beta-amyloid (Abeta) 1-42 on GSK-3beta and its major upstream regulators, intracellular calcium and protein kinases C and B (PKC and PKB) in human SH-SY5Y neuroblastoma cells. ApoE3 induced a mild, transient, Ca2+-independent and early activation of GSK-3beta. ApoE4 effects were biphasic, with an early strong GSK-3beta activation that was partially dependent on extracellular Ca2+, followed by a GSK-3beta inactivation. ApoE4 also activated PKC-alpha and PKB possibly giving the subsequent GSK-3beta inhibition. Abeta(1-42) effects were also biphasic with a strong activation dependent partially on extracellular Ca2+ followed by an inactivation. Abeta(1-42) induced an early and potent activation of PKC-alpha and a late decrease of PKB activity. ApoE4 and Abeta(1-42) were more toxic than apoE3 as shown by MTT reduction assays and generation of activated caspase-3. ApoE4 and Abeta(1-42)-induced early activation of GSK-3beta could lead to apoptosis and tau hyperphosphorylation. A late inhibition of GSK-3beta through activation of upstream kinases likely compensates the effects of apoE4 and Abeta(1-42) on GSK-3beta, the unbalanced regulation of which may contribute to AD pathology.

Caspase cleavage of exon 9 deleted presenilin-1 is an early event in apoptosis induced by calcium ionophore A 23187 in SH-SY5Y neuroblastoma cells.

Presenilins (PSs) are mutated in a majority of familial Alzheimer disease (FAD) cases. Mutated PSs may cause FAD by a number of pro-apoptotic mechanisms, or by regulating gamma-secretase activity, a protease involved in beta-amyloid precursor protein processing to the neurotoxic beta-amyloid peptide. Besides their normal endoproteolytic processing, PSs are substrates for caspases, being cleaved to alternative N-terminal and C-terminal fragments. So far little is known about the role of PSs cleavage in the apoptotic machinery. Here, we used SH-SY5Y neuroblastoma cells stably transfected with wild-type or exon 9 deleted presenilin 1 (PS1) in a time-course study after the exposure to the calcium ionophore A23187. During and after exposure to A 23187, intracellular calcium levels were higher in exon 9 deleted PS1 cells as compared with non-transfected and wild-type PS1 transfected cells. Cell death and the enrichment of apoptotic cells after A23187 exposure were increased by overexpression of exon 9 deleted PS1 as compared with the control cell lines. Wild-type PS1 cells were compared with exon 9 deleted PS1 cells and the temporal relationship between PS1 and other caspase substrates cleavages was analyzed. Exon 9 deleted PS1 cells exhibited a higher caspase-3 activation and a greater cleavage of PS1 and poly(ADP-ribose) polymerase (PARP) compared with wild-type PS1 cells. Exon 9 deleted PS1 cleavage occurred earlier than other caspase substrate cleavages (i.e., PARP and gelsolin), simultaneous with minimum detectable caspase-3 activation. Therefore, alternative cleavage of PS1 may play an important role for the regulation of the proteolytic cascade activated during apoptosis.

Regulation of apolipoprotein E secretion in rat primary hippocampal astrocyte cultures.

Apolipoprotein E isoforms may have differential effects on a number of pathological processes underlying Alzheimer's disease. Recent studies suggest that the amount, rather than the type, of apolipoprotein E may also be an important determinant for Alzheimer's disease. Therefore, understanding the regulated synthesis of apolipoprotein E is important for determining its role in Alzheimer's disease. We show here that in rat primary hippocampal astrocyte cultures, dibutyryl-cAMP increased apolipoprotein E secretion with time in a dose-dependent manner (to 177% at 48 h) and that retinoic acid potentiated this effect (to 298% at 48 h). Dibutyryl-cAMP also gave a rapid, albeit transient, increase of apolipoprotein E mRNA expression (to 200% at 1 h). In contrast, the protein kinase C activator phorbol 12-myristate 13-acetate decreased both apolipoprotein E secretion (to 59% at 48 h) and mRNA expression (to 22% at 1 h). Phorbol 12-myristate 13-acetate also reversed the effects of dibutyryl-cAMP. Apolipoprotein E secretion was also modulated by receptor agonists for the adenylyl cyclase/cAMP pathway. Isoproterenol (50 nM, a beta-adrenoceptor agonist) enhanced, while clonidine (250 nM, an alpha2-adrenoceptor agonist) decreased, secreted apolipoprotein E. We also analysed the effects of agonists for the phospholipase C/protein kinase C pathway. Arterenol (1 microM, an alpha1-adrenoceptor agonist) and serotonin (2.5 microM) enhanced, whereas carbachol (10 microM, an acetylcholine muscarinic receptor agonist) decreased secreted apolipoprotein E. The effects of these non-selective receptor agonists were modest, probably due to effects on different signalling pathways. Arterenol also potentiated the isoproterenol-mediated increase. We also show that phorbol 12-myristate 13-acetate and dibutyryl-cAMP have opposite effects on nerve growth factor, as compared to apolipoprotein E, secretion, suggesting that the results obtained were unlikely to be due to a general effect on protein synthesis. We conclude that astrocyte apolipoprotein E production can be regulated by factors that affect cAMP intracellular concentration or activate protein kinase C. Alterations in these signalling pathways in Alzheimer's disease brain may have consequences for apolipoprotein E secretion in this disorder.

Apolipoprotein E isoform-specific disruption of phosphoinositide hydrolysis: protection by estrogen and glutathione.

The mechanism(s) by which the E4 isoform of apolipoprotein E (apoE4) influences Alzheimer's disease (AD) are not fully known. We report that apoE4, but not apoE3, disrupts carbachol-stimulated phosphoinositide (PI) hydrolysis in SH-SY5Y neuroblastoma cells. Carbachol responses were also disrupted by beta-amyloid (Abeta) (1-42) and apoE4/Abeta(1-42) complexes, but not by apoE3/Abeta(1-42). Glutathione and estrogen protected against apoE4 and Abeta(1-42) effects, as well as those of H(2)O(2). Estrogen protection was partially blocked by wortmannin, suggesting the involvement of phosphatidylinositol 3-kinase. An apoE4-induced disruption of acetylcholine muscarinic receptor-mediated signalling may explain the lower effectiveness of cholinergic replacement treatments in apoE4 AD patients. Also, the beneficial effect of estrogen in AD may be partially due to its ability to protect against apoE4- and Abeta(1-42)-mediated disruption of PI hydrolysis.

Apolipoprotein E and beta A4-amyloid: signals and effects.

In humans, the apolipoprotein E gene (APOE) is polymorphic with the alleles APOE epsilon 2, 3 and 4 coding for apolipoproteins (Apo) E2, 3 and 4. Apart from age, the APOE epsilon 4 allele represents the most important risk factor in sporadic Alzheimer's disease (AD). Compared to APOE epsilon 3 homozygotes, the histopathological onset of tau pathology is found 1-2 decades earlier but progresses with the same speed. ApoE dose-dependently and specifically increases free intraneuronal calcium levels in the order ApoE4 > ApoE3 > ApoE2. This effect is amplified in the presence of beta A4-peptide. The ApoE effects on calcium are not affected by the blockade of action potentials with tetrodotoxin, or by inhibition of common ApoE binding sites. The calcium channel involved has been identified as a P/Q-type-like channel. Brain tissue ApoE levels differ with respect to APOE alleles and Braak-stage for Alzheimer-histopathology. The production of ApoE in astrocytes is controlled by several receptor/effector systems such as adrenoceptors and cAMP. In the presence of beta A4-peptide fragments, astrocytes stop their synthesis of ApoE resulting in a massive reduction in the bioavailability of ApoE. In the periphery, ApoE directs cholesterol transport and thereby influences its cellular concentrations. In neurons, changes in the concentration of cholesterol influence the phosphorylation status of the microtubule-associated protein tau at sites known to be altered in AD.

Effects of apolipoprotein E (apoE) isoforms, beta-amyloid (Abeta) and apoE/Abeta complexes on protein kinase C-alpha (PKC-alpha) translocation and amyloid precursor protein (APP) processing in human SH-SY5Y neuroblastoma cells and fibroblasts.

We investigated the effects of different apolipoprotein E (apoE) isoforms, Abeta (1-42), and apoE/Abeta complexes on PKC-alpha translocation and APP processing in human SH-SY5Y neuroblastoma cells and fibroblasts. Treatment of cells with either 10 nM apoE3 or apoE4, 10 microM Abeta (1-42), or apoE/Abeta complexes induced significant translocation of PKC-alpha in both cell types. Effects were seen using both human recombinant apoE and apoE loaded into beta-very low density lipoprotein (beta-VLDL) particles. Time course (5-24 h) studies of APP processing revealed that some conditions induced transient or moderate increases in the secretion of proteins detected by 22C11. In contrast, the secretion of alpha-secretase cleaved APP was either not modified or transiently decreased, as determined by immunoblotting with the antibody 6E10. These results suggest that apoE, Abeta (1-42) and apoE/Abeta complexes can modulate PKC activity but do not have major consequences for APP processing. These effects could contribute to the reported PKC alterations seen in AD. However, it is unlikely that the contribution of different apoE isoforms to AD pathology occurs via effects on APP processing.

Beta-VLDL protects against A beta(1-42) and apoE toxicity in human SH-SY5Y neuroblastoma cells.

The toxic effects of beta-amyloid (A beta) (1-42), apolipoprotein E (apoE) isoforms, and apoE/A beta complexes were studied in human SH-SY5Y neuroblastoma cells and fibroblasts using MTT reduction. In SH-SY5Y cells, A beta(1-42) gave time-dependent toxicity over 2-48 h, which was reduced by co-incubation with rabbit beta-very low density lipoproteins (beta-VLDL). Human recombinant apoE3 and E4 isoforms were also toxic by themselves and also potentiated A beta effects when used alone, but not when associated with beta-VLDL. None of the treatments were toxic to human fibroblasts. These results suggest that beta-VLDL has a protective role on A beta-induced neurotoxicity and that the status of apoE or the conformation of lipoprotein containing apoE particles may be important for determining the contribution of apoE to neurodegeneration.

Apolipoprotein E: a major piece in the Alzheimer's disease puzzle.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with multiple etiologies. The presence of the E4 isoform of apolipoprotein E (apoE) has been shown to increase the risk and to decrease the age of onset for AD and is the major susceptibility factor known for the disease. ApoE4 has been shown to intensify all the biochemical disturbances characteristic of AD, including beta amyloid (Abeta) deposition, tangle formation, neuronal cell death, oxidative stress, synaptic plasticity and dysfunctions of lipid homeostasis and cholinergic signalling. In contrast, other apoE isoforms are protective. Here we review and discuss these major hypotheses of the apoE4-AD association.

Nicergoline stimulates protein kinase C mediated alpha-secretase processing of the amyloid precursor protein in cultured human neuroblastoma SH-SY5Y cells.

We investigated the ability of the antidementia agents, nicergoline, aniracetam and hydergine to stimulate PKC mediated alpha-secretase amyloid precursor protein (APP) processing in cultured human neuroblastoma SH-SY5Y cells. Western immunoblotting of cell conditioned media using the Mabs 22C11 and 6E10 revealed the presence of 2 bands with molecular mass of 90 and 120 kDa, corresponding to possible alternatively glycosylated forms of secreted APP (APPs). Short-term (30 min and 2 h) treatment of cells with nicergoline gave an increased intensity of both bands, compared to non-treated cells. Maximal nicergoline effects, of the order of 150-200% over basal APPs release, were seen at concentrations between 1 and 10 microM. Under the same condition, 1 microM PdBu, used as a positive control, gave 500-1000% increases of basal APPs release. In contrast, aniracetam and hydergine, did not show any effect on APPs secretion. 2 h treatment with nicergoline had no effect on cellular full-length APP levels, as determined by immunoblotting of cell extracts with 22C11 and CT15 antibodies. Immunoblotting with PKC isoform specific antibodies of soluble and membrane fractions prepared from 2 h treated cells, showed that nicergoline (50 microM) and PdBu (1 microM) both induced translocation of PKC alpha, gamma and epsilon, but not PKC beta. The involvement of PKC in mediating nicergoline stimulated APPs release was also studied using specific inhibitors. 1 microM calphostin C, a broad range PKC inhibitor, significantly reduced both PdBu (1 microM) and nicergoline (10 microM) induced APPs release. In contrast, Go6976 (1 microM), a selective PKC alpha and beta1 inhibitor, as well as the cAMP-dependent protein kinase inhibitor, H89 (1 microM) were without effect. These results indicate that nicergoline can modulate alpha-secretase APP processing by a PKC dependent mechanism that is likely to involve the gamma and epsilon isoforms of this enzyme.

Protein kinase C and amyloid precursor protein processing in skin fibroblasts from sporadic and familial Alzheimer's disease cases.

Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimer's disease cases. In contrast, familial Alzheimer's disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.