No activation of human pregnane X receptor by hyperforin-related phloroglucinols.

The acylated phloroglucinol, hyperforin, the main active ingredient of St. John's Wort, exerts antidepressant properties via indirect inhibition of serotonin reuptake by selectively activating the canonical transient receptor potential channel 6 (TRPC6). Hyperforin treatment can lead to drug-drug interactions due to potent activation of the nuclear receptor PXR (NR1I2), a key transcriptional regulator of genes involved in drug metabolism and transport. It was previously shown that synthetic acylated phloroglucinol derivatives activate TRPC6 with similar potency as hyperforin. However, their interaction potential with PXR remained unknown. Here we investigated five synthetic TRPC6-activating phloroglucinol derivatives and four TRPC6-nonactivating compounds compared with hyperforin and rifampicin for their potential to activate PXR in silico and in vitro. Computational PXR pharmacophore modeling did not indicate potent agonist or antagonist interactions for the TRPC6-activating derivatives, whereas one of them was suggested by docking studies to show both agonist and antagonist interactions. Hyperforin and rifampicin treatment of HepG2 cells cotransfected with human PXR expression vector and a CYP3A4 promoter-reporter construct resulted in potent PXR-dependent induction, whereas all TRPC6-activating compounds failed to show any PXR activation or to antagonize rifampicin-mediated CYP3A4 promoter induction. Hyperforin and rifampicin treatment of primary human hepatocytes resulted in highly correlated induction of PXR target genes, whereas treatment with the phloroglucinol derivatives elicited moderate gene expression changes that were only weakly correlated with those of rifampicin and hyperforin treatment. These results show that TRPC6-activating phloroglucinols do not activate PXR and should therefore be promising new candidates for further drug development.

TRPC6 channel-mediated neurite outgrowth in PC12 cells and hippocampal neurons involves activation of RAS/MEK/ERK, PI3K, and CAMKIV signaling.

The non-selective cationic transient receptor canonical 6 (TRPC6) channels are involved in synaptic plasticity changes ranging from dendritic growth, spine morphology changes and increase in excitatory synapses. We previously showed that the TRPC6 activator hyperforin, the active antidepressant component of St. John's wort, induces neuritic outgrowth and spine morphology changes in PC12 cells and hippocampal CA1 neurons. However, the signaling cascade that transmits the hyperforin-induced transient rise in intracellular calcium into neuritic outgrowth is not yet fully understood. Several signaling pathways are involved in calcium transient-mediated changes in synaptic plasticity, ranging from calmodulin-mediated Ras-induced signaling cascades comprising the mitogen-activated protein kinase, PI3K signal transduction pathways as well as Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) and CAMKIV. We show that several mechanisms are involved in TRPC6-mediated synaptic plasticity changes in PC12 cells and primary hippocampal neurons. Influx of calcium via TRPC6 channels activates different pathways including Ras/mitogen-activated protein kinase/extracellular signal-regulated kinases, phosphatidylinositide 3-kinase/protein kinase B, and CAMKIV in both cell types, leading to cAMP-response element binding protein phosphorylation. These findings are interesting not only in terms of the downstream targets of TRPC6 channels but also because of their potential to facilitate further understanding of St. John's wort extract-mediated antidepressant activity. Alterations in synaptic plasticity are considered to play an important role in the pathogenesis of depression. Beside several other proteins, TRPC6 channels regulate synaptic plasticity. This study demonstrates that different pathways including Ras/MEK/ERK, PI3K/Akt, and CAMKIV are involved in the improvement of synaptic plasticity by the TRPC6 activator hyperforin, the antidepressant active constituent of St. John's wort extract.

Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca(2+) -permeable TRPC6 channels.

The standardized extract of the St. John's wort plant (Hypericum perforatum) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na(+) concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca(2+) -permeable, resulting in intracellular Ca(2+) elevations. Indeed, hyperforin activates TRPC6-mediated currents and Ca(2+) transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca(2+) transients and depolarizing inward currents sensitive to the TRPC channel blocker La(3+) , thus resembling the actions of the neurotrophin brain-derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF.

Improvement of mitochondrial function and dynamics by the metabolic enhancer piracetam.

The metabolic enhancer piracetam is used in many countries to treat cognitive impairment in aging, brain injuries, as well as dementia such as AD (Alzheimer's disease). As a specific feature of piracetam, beneficial effects are usually associated with mitochondrial dysfunction. In previous studies we were able to show that piracetam enhanced ATP production, mitochondrial membrane potential as well as neurite outgrowth in cell and animal models for aging and AD. To investigate further the effects of piracetam on mitochondrial function, especially mitochondrial fission and fusion events, we decided to assess mitochondrial morphology. Human neuroblastoma cells were treated with the drug under normal conditions and under conditions imitating aging and the occurrence of ROS (reactive oxygen species) as well as in stably transfected cells with the human wild-type APP (amyloid precursor protein) gene. This AD model is characterized by expressing only 2-fold more human Aß (amyloid ß-peptide) compared with control cells and therefore representing very early stages of AD when Aß levels gradually increase over decades. Interestingly, these cells exhibit an impaired mitochondrial function and morphology under baseline conditions. Piracetam is able to restore this impairment and shifts mitochondrial morphology back to elongated forms, whereas there is no effect in control cells. After addition of a complex I inhibitor, mitochondrial morphology is distinctly shifted to punctate forms in both cell lines. Under these conditions piracetam is able to ameliorate morphology in cells suffering from the mild Aß load, as well as mitochondrial dynamics in control cells.

Studies on the enantiomers of RC-33 as neuroprotective agents: isolation, configurational assignment, and preliminary biological profile.

In this study we addressed the role of chirality in the biological activity of RC-33, recently studied by us in its racemic form. An asymmetric synthesis procedure was the first experiment, leading to the desired enantioenriched RC-33 but with an enantiomeric excess (ee) not good enough for supporting the in vitro investigation. An enantioselective high-performance liquid chromatography (HPLC) procedure was then successfully carried out, yielding both RC-33 enantiomers in amounts and optical purity suitable for the pharmacological study. The absolute configuration of pure enantiomers was easily assigned exploiting the asymmetric synthesis previously devised. As emerged in the preliminary in vitro biological investigation, (S)- and (R)-RC-33 possess a comparable affinity towards the σ1 receptor and a very a similar behavior in the calcium influx assay, resulting in an equally effective σ1 receptor agonist. Overall, the results obtained so far suggest that the interaction with the biological target is nonstereoselective and leads us to hypothesize that there is a lack of stereoselectivity in the biological activity of RC-33.

Laser-induced liquid bead ion desorption mass spectrometry: an approach to precisely monitor the oligomerization of the ß-amyloid peptide.

In the present work, the recently developed laser-induced liquid bead ion desorption mass spectrometry (LILBID MS) is applied as a novel technique to study Aß oligomerization, thought to be crucial in Alzheimer's disease (AD). The characterization of the earliest nucleation events of this peptide necessitates the application of several techniques to bridge the gap between small oligomers and large fibrils. We precisely monitored in time the transformation of monomeric Aß (1-42) into oligomeric Aß(n) (n < 20) and its dependence on concentration and agitation. The distribution shows signs of the hexamer being crucial in the assembly process. The intensity of the monomer decreases in time with a time constant of about 9 h. After a lag time of around 10 h, a phase transition occurred in which the total ion current of the oligomers goes to nearly zero. In this late stage of aggregation, protofibrils are formed and mass spectrometry is no longer sensitive. Here fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM) are complementary tools for detection and size characterization of these large species. We also utilized the oligomers of Aß (1-42) as a model of the corresponding in vivo process to screen the efficacy and specificity of small molecule inhibitors of oligomerization. The LILBID results are in excellent agreement with condensed phase behavior determined in other studies. Our data identified LILBID MS as a powerful technique that will advance the understanding of peptide oligomerization in neurodegenerative diseases and represents a powerful tool for the identification of small oligomerization inhibitors.

Effects of the standardized Ginkgo biloba extract EGb 761® on neuroplasticity.

Neuroplasticity, the ability of synapses to undergo structural adaptations in response to functional demand or dysfunctions is increasingly impaired in aging and Alzheimer's disease. EGb761® has been shown in several preclinical reports to increase nearly all aspects of impaired neuroplasticity (long-term potentiation, spine density, neuritogenesis, neurogenesis). While all three fractions of constituents (ginkgolides, flavonoids, bilobalide) seem to be active, the flavonoids and specifically the aglycone isorhamnetin seem to be most relevant.

Treatment duration (persistence) of basal insulin supported oral therapy (BOT) in Type-2 diabetic patients: comparison of insulin glargine with NPH insulin.

OBJECTIVE: To compare the persistence (treatment duration) of basal insulin supported oral therapy (BOT) using insulin glargine (GLA) or NPH insulin (NPH) in Type-2 diabetic patients. METHODS: This retrospective cohort study reports results from an analysis of claims data from prescriptions for ambulatory patients within the German Statutory Health Insurance scheme. The study is based on claims data from more than 80% of German community pharmacies. Treatment duration until switching to a basal bolus treatment regimen (intensified conventional insulin therapy: ICT) was determined in insulin-naïve patients who began treatment with BOT using GLA or NPH between 01/2003 and 12/2006. RESULTS: A total of 97,998 patients (61,070 GLA and 36,928 NPH) were included. Within the observation period, 23.5% of GLA patients and 28.0% of NPH patients switched from BOT to ICT. The upper quartile of probability of continuation of therapy (the 75th percentile) was reached after 769 days in GLA patients and after 517 days in NPH patients. Therefore, the risk of switching to ICT was significantly higher with NPH compared to GLA: hazard ratios were 1.34 (99% CI: 1.29-1.38; unadjusted) and 1.22 (99% CI: 1.18-1.27) after adjustment for predefined covariates. Various sensitivity analyses using modified inclusion criteria and endpoint definitions were applied and these confirmed the initial results. CONCLUSION: Type-2 diabetic patients under BOT with GLA stayed significantly longer on the initial therapy before switching to ICT than patients on BOT using NPH.

Ginkgo biloba extract (EGb761) influences monoaminergic neurotransmission via inhibition of NE uptake, but not MAO activity after chronic treatment.

In order to explain cognition-enhancing effects of standardized Ginkgo biloba extract (EGb761), an increase of central monoaminergic neurotransmission has been suggested, but the underlying mechanisms have not yet been elucidated. Here, we confirm that the norepinephrine (NET), the serotonin (SERT), the dopamine (DAT) uptake transporters and MAO activity are inhibited by EGb761 in vitro, although rather high concentrations are required for inhibition of MAO-A and MAO-B activity. However, after 14 days of daily oral treatment with 100mg/kg EGb761 only NE uptake is significantly decreased in NMRI mice, while 5-HT uptake and MAO activity are not affected. As synaptic dopamine clearance in the frontal cortex is mediated by NET, not DAT, these findings may give an explanation for the enhancement of dopaminergic neurotransmission by EGb761 seen in animal models, presumably linked to its positive effects on cognition and attention.

The amyloid precursor protein potentiates CHOP induction and cell death in response to ER Ca2+ depletion.

Here we investigated the role of the amyloid precursor protein (APP) in regulation of Ca(2+) store depletion-induced neural cell death. Ca(2+) store depletion from the endoplasmic reticulum (ER) was induced by the SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) inhibitor thapsigargin which led to a rapid induction of the unfolded protein response (UPR) and a delayed activation of executioner caspases in the cultures. Overexpression of APP potently enhanced cytosolic Ca(2+) levels and cell death after ER Ca(2+) store depletion in comparison to vector-transfected controls. GeneChip and RT-PCR analysis revealed that the expression of classical UPR chaperone genes was not altered by overexpression of APP. Interestingly, the induction of the ER stress-responsive pro-apoptotic transcription factor CHOP was significantly upregulated in APP-overexpressing cells in comparison to vector-transfected controls. Chelation of intracellular Ca(2+) with BAPTA-AM revealed that enhanced CHOP expression after store depletion occurred in a Ca(2+)-dependent manner in APP-overexpressing cells. Prevention of CHOP induction by BAPTA-AM and by RNA interference was also able to abrogate the potentiating effect of APP on thapsigargin-induced apoptosis. Application of the store-operated channel (SOC)-inhibitors SK & F96365 and 2-APB downmodulated APP-triggered potentiation of cytosolic Ca(2+) levels and apoptosis after treatment with thapsigargin. Our data demonstrate that APP significantly modulates Ca(2+) store depletion-induced cell death in a SOC- and CHOP-dependent manner, but independent of the UPR.

Hyperforin--a key constituent of St. John's wort specifically activates TRPC6 channels.

Hyperforin, a bicyclic polyprenylated acylphloroglucinol derivative, is the main active principle of St. John's wort extract responsible for its antidepressive profile. Hyperforin inhibits the neuronal serotonin and norepinephrine uptake comparable to synthetic antidepressants. In contrast to synthetic antidepressants directly blocking neuronal amine uptake, hyperforin increases synaptic serotonin and norepinephrine concentrations by an indirect and yet unknown mechanism. Our attempts to identify the molecular target of hyperforin resulted in the identification of TRPC6. Hyperforin induced sodium and calcium entry as well as currents in TRPC6-expressing cells. Sodium currents and the subsequent breakdown of the membrane sodium gradients may be the rationale for the inhibition of neuronal amine uptake. The hyperforin-induced cation entry was highly specific and related to TRPC6 and was suppressed in cells expressing a dominant negative mutant of TRPC6, whereas phylogenetically related channels, i.e., TRPC3 remained unaffected. Furthermore, hyperforin induces neuronal axonal sprouting like nerve growth factor in a TRPC6-dependent manner. These findings support the role of TRPC channels in neurite extension and identify hyperforin as the first selective pharmacological tool to study TRPC6 function. Hyperforin integrates inhibition of neurotransmitter uptake and neurotrophic property by specific activation of TRPC6 and represents an interesting lead-structure for a new class of antidepressants.

Mitochondrial dysfunction: the first domino in brain aging and Alzheimer's disease?

With the increasing average life span of humans and with decreasing cognitive function in elderly individuals, age-related cognitive disorders including dementia have become a major health problem in society. Aging-related mitochondrial dysfunction underlies many common neurodegenerative disorders diseases, including Alzheimer's disease (AD). AD is characterized by two major histopathological hallmarks, initially intracellular and with the progression of the disease extracellular accumulation of oligomeric and fibrillar beta-amyloid (Abeta) peptides and intracellular neurofibrillary tangles (NFT) composed of hyperphosphorylated tau protein. In this review, the authors focus on the latest findings in AD animal models indicating that these histopathological alterations induce deficits in the function of the complexes of the respiratory chain and therefore consecutively result in mitochondrial dysfunction. This parameter is intrinsically tied to oxidative stress. Both are early events in aging and especially in the pathogenesis of aging-related severe neurodegeneration. Ginkgo biloba extract seems to be of therapeutic benefit in the treatment of mild to moderate dementia of different etiology, although the data are quite heterogeneous. Herein, the authors suggest that mitochondrial protection and subsequent reduction of oxidative stress are important components of the neuroprotective activity of Ginkgo biloba extract.

Functionally selective dopamine D2, D3 receptor partial agonists.

Dopamine D2 receptor-promoted activation of Gα(o) over Gα(i) may increase synaptic plasticity and thereby might improve negative symptoms of schizophrenia. Heterocyclic dopamine surrogates comprising a pyrazolo[1,5-a]pyridine moiety were synthesized and investigated for their binding properties when low- to subnanomolar K(i) values were determined for D(2L), D(2S), and D3 receptors. Measurement of [(35)S]GTPγS incorporation at D(2S) coexpressed with G-protein subunits indicated significant bias for promotion of Gα(o1) over Gα(i2) coupling for several test compounds. Functionally selective D(2S) activation was most striking for the carbaldoxime 8b (Gα(o1), pEC50 = 8.87, E(max) = 65%; Gα(i2), pEC50 = 6.63, E(max) = 27%). In contrast, the investigated 1,4-disubstituted aromatic piperazines (1,4-DAPs) behaved as antagonists for ß-arrestin-2 recruitment, implying significant ligand bias for G-protein activation over ß-arrestin-2 recruitment at D(2S) receptors. Ligand efficacy and selectivity between D(2S) and D3 activation were strongly influenced by regiochemistry and the nature of functional groups attached to the pyrazolo[1,5-a]pyridine moiety.

A cell model for the initial phase of sporadic Alzheimer's disease.

Recent data suggest that the combined effect of oxidative stress due to aging and slightly elevated amyloid-ß (Aß) levels initiate Alzheimer's disease (AD) long before the clinical onset. Investigations of this early phase are hampered by the lack of cellular or animal models reflecting this scenario. We used SH-SY5Y cells stably transfected with an additional copy of the human AßPP gene and artificial aging by complex I inhibition. These cells show slightly elevated Aß levels, moderately decreased ATP levels, impaired mitochondrial membrane potential, and decreased mitochondrial respiration. Assessing mitochondrial dynamics with three different methods reveals a distinct shift toward mitochondrial fission and fragmentation in SH-SY5Y AßPPwt cells. We also performed electron cryo-tomography of isolated mitochondria to reveal that there were no major differences between SH-SY5Y control and SH-SY5Y AßPPwt mitochondria with respect to swelling or loss of cristae. Dystrophic neurites are an early pathological feature of AD. Interestingly, SH-SY5Y AßPPwt cells exhibit significantly longer neurites, likely due to substantially elevated levels of sAßPPα. Complex I inhibition also shows substantial effects on mitochondrial dynamics, impairs neuritogenesis, and elevates Aß levels in both cell types. In SH-SY5Y AßPPwt cells, these defects were more pronounced due to a relatively elevated Aß and a reduced sAßPPα production. Our findings suggest that the progression from low Aß levels to the beginning of AD takes place in the presence of oxidative stress during normal aging. This mechanism not only results from additive effects of both mechanisms on mitochondrial function but might also be additionally aggravated by altered amyloidogenic processing.

Mitochondrial dysfunction: cause and consequence of Alzheimer's disease.

The etiology of common, nonfamiliar late-onset Alzheimer's disease (LOAD) is only partly understood and seems to be extremely complex including many genetic and environmental factors. The most important environmental risk factor to develop LOAD is aging itself. Aging and LOAD are considered to be strongly linked to mitochondrial dysfunction and enhanced oxidative stress. In this review, we focus on the interaction between mitochondrial dysfunction in aging especially on defects of the respiratory chain of the oxidative phosphorylation system resulting in enhanced oxidative stress and the interplay between aging-associated mitochondrial defects and LOAD-associated mitochondrial failure. The deleterious effects of the two hallmarks of LOAD, amyloid beta, and hyperphosphorylated tau, on mitochondrial function, movement, and morphology are described as well as the toxic effects of the most relevant genetic risk factor of LOAD, the apolipoprotein E4 allele. Finally, the review provides an overview about drugs and nutritional ingredients which improve mitochondrial function or/and act as antioxidants and discusses their potential role in the treatment of LOAD.

Mitochondrial membrane fluidity is consistently increased in different models of Huntington disease: restorative effects of olesoxime.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT). One prominent target of the mutant huntingtin protein (mhtt) is the mitochondrion, affecting its morphology, distribution, and function. Thus, mitochondria have been suggested as potential therapeutic targets for the treatment of HD. Olesoxime, a cholesterol-like compound, promotes motor neuron survival and neurite outgrowth in vitro, and its effects are presumed to occur via a direct interaction with mitochondrial membranes (MMs). We examined the properties of MMs isolated from cell and animal models of HD as well as the effects of olesoxime on MM fluidity and cholesterol levels. MMs isolated from brains of aged Hdh Q111/Q111 knock-in mice showed a significant decrease in 1,6-diphenyl-hexatriene (DPH) anisotropy, which is inversely correlated with membrane fluidity. Similar increases in MM fluidity were observed in striatal STHdh Q111/Q111 cells as well as in MMs isolated from brains of BACHD transgenic rats. Treatment of STHdh cells with olesoxime decreased the fluidity of isolated MMs. Decreased membrane fluidity was also measured in olesoxime-treated MMs isolated from brains of HD knock-in mice. In both models, treatment with olesoxime restored HD-specific changes in MMs. Accordingly, olesoxime significantly counteracted the mhtt-induced increase in MM fluidity of MMs isolated from brains of BACHD rats after 12 months of treatment in vivo, possibly by enhancing MM cholesterol levels. Thus, olesoxime may represent a novel pharmacological tool to treat mitochondrial dysfunction in HD.

Adherence to antihypertensives: feasibility of two self-report instruments to investigate medication-taking behaviour in German community pharmacies.

OBJECTIVE: To design and test the feasibility of two questionnaires in German community pharmacies exploring self-reported adherence to antihypertensives. METHODS: Two self-report questionnaires were designed for patients treated with antihypertensives. The 29-item-questionnaire (long form, LF) was completed by pharmacists interviewing patients who were on the premises filling a prescription. The short form (SF; 19 items) was sent by pharmacies to patients via mail. The acceptance of the instruments by patients and pharmacists as well as the feasibility to measure medication-taking behaviour was investigated. Adherence was investigated by using a modified 5-(LF) or 6-item (SF) Morisky score. RESULTS: Of 44 community pharmacies contacted, 18 agreed to participate. Patients' response rates were 428/915 (46.8%) for the SF and 249/760 (32.8%) for the LF. One hundred and seventy-nine patients (41.8%) and 70 patients (28.1%) reported adherence problems according to the SF and LF respectively. CONCLUSIONS: To our knowledge, this is the first attempt to develop a self-report instrument for the detection of non-adherence in patients taking antihypertensives in this setting in Germany. Patients were willing to provide detailed information about their medication-taking behaviour. Underestimation of non-adherence may be more pronounced when applying the questionnaire in the pharmacy.