Targeting repulsive guidance molecule A to promote regeneration and neuroprotection in multiple sclerosis.

Repulsive guidance molecule A (RGMa) is a potent inhibitor of neuronal regeneration and a regulator of cell death, and it plays a role in multiple sclerosis (MS). In autopsy material from progressive MS patients, RGMa was found in active and chronic lesions, as well as in normal-appearing gray and white matter, and was expressed by cellular meningeal infiltrates. Levels of soluble RGMa in the cerebrospinal fluid were decreased in progressive MS patients successfully treated with intrathecal corticosteroid triamcinolone acetonide (TCA), showing functional improvements. In vitro, RGMa monoclonal antibodies (mAbs) reversed RGMa-mediated neurite outgrowth inhibition and chemorepulsion. In animal models of CNS damage and MS, RGMa antibody stimulated regeneration and remyelination of damaged nerve fibers, accelerated functional recovery, and protected the retinal nerve fiber layer as measured by clinically relevant optic coherence tomography. These data suggest that targeting RGMa is a promising strategy to improve functional recovery in MS patients.

Establishment of a secondary screening assay for P/Q-type calcium channel blockers.

Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically overactive channels. We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steady-state inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast, inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch clamp recordings. Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and--if necessary--a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable for the generation of assays in other ion channel programs.

Development of disease-modifying treatment of schizophrenia.

Development of disease-modifying therapies requires an innovative approach to drug development where novel drugs are designed to target mechanisms of interest rather than to produce preclinical effects similar to those of currently used antipsychotics. Application of such novel strategy will undoubtedly require a very deep understanding of the disease biology that is just starting to emerge. Alternatively, one may let environmental experiences of the diseased individual guide the repair process and use drugs only to facilitate the effects of experience. Such an approach would bring together functional experience that is age-, environment- and disease-dependent with the plasticity resources that may otherwise not be available. There are currently no preclinical drug-environment interaction models that can be claimed to have significant degrees of validity. Therefore, from a drug development perspective, principles that combine acute symptomatic and disease-modifying properties are clearly preferred. The question arises then how such treatments can be differentiated from those that have only symptomatic effects (i.e., most currently used antipsychotic medications). One expectation is that the former will show superior and broader efficacy (especially with longer treatment duration). Another possibility is that disease-modifying drugs will be particularly useful at the very earliest stages of the disease. Society and medical communities may not be ready yet to initiate the treatment as early as during the prodromal phase, but the situation may change by the time the science advances enough to bring a convincing case of a drug with disease-modification potential.

Development and validation of a fluorescence-based HTS assay for the identification of P/Q-type calcium channel blockers.

Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is evidence that migraine, Alzheimer's disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of disease symptoms. Although the medical need is high, there are no such compounds on the market. Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type calcium channel under control of a tetracycline (Tet) promoter. The accessory ß1.1 and α2δ1 subunits were co-expressed constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 µM. Hit-to-lead (HTL) efforts identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized. An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp analysis at inactivated channel state. The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological diseases.

Generation and therapeutic efficacy of highly oligomer-specific beta-amyloid antibodies.

Oligomers of the beta-amyloid (Abeta) peptide have been indicated in early neuropathologic changes in Alzheimer's disease. Here, we present a synthetic Abeta(20-42) oligomer (named globulomer) with a different conformation to monomeric and fibrillar Abeta peptide, enabling the generation of highly Abeta oligomer-specific monoclonal antibodies. The globulomer-derived antibodies specifically detect oligomeric but not monomeric or fibrillar Abeta in various Abeta preparations. The globulomer-specific antibody A-887755 was able to prevent Abeta oligomer binding and dynamin cleavage in primary hippocampal neurons and to reverse globulomer-induced reduced synaptic transmission. In amyloid precursor protein (APP) transgenic mice, vaccination with Abeta globulomer and treatment with A-887755 improved novel object recognition. The cognitive improvement is likely attributable to reversing a deficit in hippocampal synaptic spine density in APP transgenic mice as observed after treatment with A-887755. Our findings demonstrate that selective reduction of Abeta oligomers by immunotherapy is sufficient to normalize cognitive behavior and synaptic deficits in APP transgenic mice.

Effects of a positive allosteric modulator of group II metabotropic glutamate receptors, LY487379, on cognitive flexibility and impulsive-like responding in rats.

Orthosteric group II metabotropic glutamate receptor (mGluR) agonists are regarded as novel, effective medications for all major symptom domains of schizophrenia, including cognitive disturbances. mGluR2s also can be affected in a more subtle way by positive allosteric modulators (PAMs) characterized by a unique degree of subtype selectivity and neuronal frequency-dependent activity. Because currently available treatments for schizophrenia do not improve cognitive dysfunction, the main aim of the present study was to examine the effects of a mGluR2 PAM, N-(4-(2-methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379), on rat cognitive flexibility and impulsive-like responding, assessed in an attentional set-shifting task (ASST) and a differential reinforcement of low-rate 72 s (DRL72) schedule of food reinforcement. In addition, in vivo microdialysis was used to assess the drug's impact on cortical levels of dopamine, norepinephrine, serotonin, and glutamate. Rats treated with LY487379 (30 mg/kg) required significantly fewer trials to criteria during the extradimensional shift phase of the ASST. Under a DRL72 schedule, LY487379 (30 mg/kg) decreased the response rate and increased the number of reinforcers obtained. These effects were accompanied by the shift of the frequency distribution of responses toward longer inter-response time durations. LY487379 significantly enhanced extracellular norepinephrine and serotonin levels in the medial prefrontal cortex. In summary, the present study demonstrates that a mGluR2 PAM, LY487379, promotes cognitive flexibility and facilitates behavioral inhibition. These procognitive effects may contribute to the therapeutic efficacy of agents stimulating mGluR2 in schizophrenia.

Calpain inhibition prevents amyloid-beta-induced neurodegeneration and associated behavioral dysfunction in rats.

Amyloid-beta (Abeta) is toxic to neurons and such toxicity is - at least in part - mediated via the NMDA receptor. Calpain, a calcium dependent cystein protease, is part of the NMDA receptor-induced neurodegeneration pathway, and we previously reported that inhibition of calpain prevents excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. The present study reveals that inhibition of calpain is also neuroprotective in an in vivo model of Abeta oligomer-induced neurodegeneration in rats. Abeta-induced lesions of the nucleus basalis induced a significant decrease in the number of cholinergic neurons and their projecting fibers, as determined by analysis of choline-acetyltransferase in the nucleus basalis magnocellularis and cortical mantle of the lesioned animals. Treatment with the calpain inhibitor A-705253 significantly attenuated cholinergic neurodegeneration in a dose-dependent manner. Calpain inhibition also significantly diminished the accompanying neuroinflammatory response, as determined by immunohistochemical analysis of microglia activation. Administration of beta-amyloid markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, A-705253, dose-dependently prevented this behavioral deficit. In order to determine whether pre-treatment with the calpain inhibitor is necessary to exhibit its full protective effect on neurons we induced Abeta toxicity in primary neuronal cultures and administered A-705253 at various time points before and after Abeta oligomer application. Although the protective effect was higher when A-705253 was applied before induction of Abeta toxicity, calpain inhibition was still beneficial when applied up to 1h post-treatment. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of Abeta oligomer-induced neuronal decline and associated cognitive deterioration.

Glutamatergic (N-methyl-D-aspartate receptor) hypofrontality in schizophrenia: too little juice or a miswired brain?

Dopamine D2 receptor blockade has been an obligate mechanism of action present in all medications that effectively treat positive symptoms of schizophrenia (e.g., delusions and hallucinations) and have been approved by regulatory agencies since the 1950s. Blockade of 5-hydroxytryptamine(2A) receptors plays a contributory role in the actions of the second generation of antipsychotic drugs, the so-called atypical antipsychotics. Nevertheless, substantial unmet medical needs remain for the treatment of negative symptoms and cognitive dysfunction. Recognition that dissociative anesthetics block the N-methyl-D-aspartate (NMDA) receptor channel has inspired a search for glutamatergic therapeutic mechanisms because ketamine and phencyclidine are known to induce psychotic-like symptoms in healthy volunteers and exacerbate the symptoms of patients with schizophrenia. Current pathophysiological theories of schizophrenia emphasize that hypofunction of NMDA receptors at critical sites in local circuits modulate the function of a given brain region or control projections from one region to another (e.g., hippocampal-cortical or thalamocortical projections). The demonstration that a metabotropic glutamate 2/3 (mGlu2/3) receptor agonist prodrug decreased both positive and negative symptoms of schizophrenia raised hopes that glutamatergic mechanisms may provide therapeutic advantages. In addition to discussing the activation of mGlu2 receptors with mGlu2/3 receptor agonists or mGlu2 receptor positive allosteric modulators (PAMs), we discuss other methods that may potentially modulate circuits with hypofunctional NMDA receptors such as glycine transporter inhibitors and mGlu5 receptor PAMs. The hope is that by modulating glutamatergic neurotransmission, the dysfunctional circuitry of the schizophrenic brain (both local circuits and long-loop pathways) will be improved.

Amphetamine decreases behavioral inhibition by stimulation of dopamine D2, but not D3, receptors.

Behavioral disinhibition is a manifestation of impulsive behavior that is prominent in the psychopathology of various psychiatric disorders such as addiction, attention-deficit hyperactivity disorder, mania, and personality disorders. Impulsivity may be studied by measuring anticipatory responses made before the presentation of a food-predictive, brief light stimulus in a two-choice serial reaction time task. In such serial reaction time tasks, amphetamine has been shown to produce dose-dependent increases in premature responding in a manner dependent on dopamine D(2)-like receptor stimulation. So far, it is unknown whether it is the D(2) or D(3) receptor that is involved in this form of impulsivity. In this study, rats were trained in a two-choice serial reaction time task until baseline performance was stable. Next, effects of the dopamine D(2) preferring antagonist L-741,626 and selective D(3) antagonist SB-277011 were assessed alone and in the presence of amphetamine. Neither L-741,626 nor SB-277011 affected behavioral inhibition, although the latter significantly increased reaction time at 10 mg/kg. Amphetamine dose-dependently increased impulsivity. The effect of amphetamine was attenuated by L-741,626 (3 mg/kg), whereas SB-277011 (3 mg/kg) had no effect. Therefore, amphetamine-induced behavioral disinhibition depends on D(2), but not D(3), receptor stimulation.

Improvement of an acid phosphatase/DHAP-dependent aldolase cascade reaction by using directed evolution.

To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.

Complementary chemoenzymatic routes to both enantiomers of febrifugine.

Two complementary strategies for the synthesis of febrifugine are detailed based on previously developed chemoenzymatic approaches to the 3-hydroxypiperidine skeleton. The introduction of the quinazolone-containing side chain in both strategies was based on an N-acyliminium ion-mediated coupling reaction.

Stimulating neuroregeneration as a therapeutic drug approach for traumatic brain injury.

Traumatic brain injury, a silent epidemic of modern societies, is a largely neglected area in drug development and no drug is currently available for the treatment of patients suffering from brain trauma. Despite this grim situation, much progress has been made over the last two decades in closely related medical indications, such as spinal cord injury, giving rise to a more optimistic approach to drug development in brain trauma. Fundamental insights have been gained with animal models of central nervous system (CNS) trauma and spinal cord injury. Neuroregenerative drug candidates have been identified and two of these have progressed to clinical development for spinal cord injury patients. If successful, these drug candidates may be used to treat brain trauma patients. Significant progress has also been made in understanding the fundamental molecular mechanism underlying irreversible axonal growth arrest in the injured CNS of higher mammals. From these studies, we have learned that the axonal retraction bulb, previously regarded as a marker for failure of regenerative growth, is not static but dynamic and, therefore, amenable to pharmacotherapeutic approaches. With the development of modified magnetic resonance imaging methods, fibre tracts can be visualised in the living human brain and such imaging methods will soon be used to evaluate the neuroregenerative potential of drug candidates. These significant advances are expected to fundamentally change the often hopeless situation of brain trauma patients and will be the first step towards overcoming the silent epidemic of brain injury.

Running wheel activity is sensitive to acute treatment with selective inhibitors for either serotonin or norepinephrine reuptake.

RATIONALE: Most antidepressants (AD) directly or indirectly enhance the serotonergic tone in the CNS. Since the serotonin system is involved in both, the modulation of mood and motor behavior, it was reasoned that these drugs might also interfere with running wheel activity (RWA), a form of positively motivated motor behavior, which might be linked to pathological states like obsessive-compulsive disorder (OCD). OBJECTIVES: We used RWA to characterize ADs from all major classes. Effects on RWA were compared to effects on general locomotor activity (LOC) to control for unspecific effects on general locomotion. METHODS: Two hours before lights-off, mice were treated with either vehicle or one of the following AD: the selective serotonin reuptake inhibitors (SSRIs) citalopram (3-10 mg/kg), paroxetine (1-10 mg/kg) and fluoxetine (2-6.6 mg/kg), the selective norepinephrine reuptake inhibitor (SNRI) reboxetine (1-10 mg/kg), the monoamine oxidase (MAO) inhibitors tranylcypromine (1-3 mg/kg) and moclobemide (3-10 mg/kg), and the tricyclic ADs desipramine and imipramine (10-30 mg/kg, each). LOC and RWA were measured after lights-off. RESULTS: At the highest dose tested, all ADs, with the exception of the MAO inhibitors, significantly reduced RWA. Both tricyclics inhibited RWA only at doses that similarly affected LOC. In contrast, all SSRI and reboxetine inhibited RWA at doses that left LOC unaffected. CONCLUSIONS: SSRI and the SNRI reboxetine inhibit RWA at doses not suppressing LOC. RWA may represent a simple behavioral readout of positively motivated behavior that merits further attention for psychopharmacology.

Inhibitors of GlyT1 affect glycine transport via discrete binding sites.

In the forebrain, synaptic glycine concentrations are regulated through the glycine transporter GlyT1. Because glycine is a coagonist of the N-methyl-D-aspartate (NMDA) receptor (NMDAR), which has been implicated in schizophrenia, inhibition of GlyT1 is thought to provide an option for the treatment of schizophrenia. In support of this hypothesis, GlyT1 inhibitors facilitate in vivo NMDAR function and demonstrate antipsychotic-like effects in animal models. Among the specific GlyT1 inhibitors, substituted N-methyl-glycine (sarcosine) derivatives (e.g., (R)-N[3-(4'fluorophenyl)-3-(4'phenyl-phenoxy)propyl]-sarcosine [NFPS], (R)-N[3-phenyl-3-(4'-(4-toluoyl)phenoxy)-propyl]sarcosine [(R)-NPTS], and (R,S)-(+/-)N-methyl-N-[(4-trifluoromethyl)phenoxy]-3-phenyl-propylglycine [Org24589]), and non-sarcosine-containing inhibitors, such as 2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride (SSR504734), have been described. In the present study, we analyzed the mode of interaction of these compounds with GlyT1 by using electrophysiological measurements in Xenopus laevis oocytes, and with two binding assays, using [(3)H](R)-NPTS or 2-chloro-N-[(S)-phenyl[(2S)-N-methylpiperidin-2-yl]-methyl]-3-trifluoromethyl benzamide monohydrochloride ([(3)H]N-methyl-SSR504734) as radioligands. Inhibition of electrogenic glycine transport by sarcosine-based compounds was apparently irreversible and independent of glycine concentration. The latter indicates a noncompetitive mode of action. In contrast, both SSR504734 and N-methyl-SSR504734 exhibited reversible and competitive inhibition of glycine transport. In GlyT1-expressing membranes, the binding of the novel radioligand [(3)H]N-methyl-SSR504734 to a single site on GlyT1 was competitively displaced by glycine and SSR504734 but noncompetitively by sarcosine-based compounds. Inversely, [(3)H](R)-NPTS binding was competitively inhibited by sarcosine-based compounds, whereas glycine, SSR504734, and N-methyl-SSR504734 noncompetitively decreased maximal binding. Our data indicate that besides exerting an apparently irreversible or reversible inhibition, GlyT1 inhibitors differ by exhibiting either a noncompetitive or competitive mode of inhibition. The divergent modes of inhibition may significantly affect the efficacy and tolerability of these drugs.

Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction.

N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cysteine protease, has been identified as part of such an NMDA receptor-induced excitotoxic signaling pathway. The present study addressed the question of whether inhibition of calpain can prevent neuronal cell death and associated behavioral deficits in a disease-relevant animal model, which is based on excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. Excitotoxic lesions of the nucleus basalis with NMDA induced a markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide (A-705253), dose-dependently prevented the behavioral deficit. Subsequent analysis of choline acetyltransferase in the cortical mantle of the lesioned animals revealed that application of A-705253 dose-dependently and significantly attenuated cholinergic neurodegeneration. Calpain inhibition also significantly diminished the accompanying gliosis, as determined by immunohistochemical analysis of microglia activation. Finally, inhibition of calpain by A-705253 and the peptidic calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO did not impair long-term potentiation in hippocampal slices, indicating that calpain inhibition interrupts NMDA excitotoxicity pathways without interfering with NMDA receptor-mediated signaling involved in cognition. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of excitotoxicity-induced neuronal decline without interfering with the physiological neuronal functions associated with learning and memory processes. Thus, calpain inhibition may be a promising and novel approach for the treatment of various neurodegenerative disorders.

N,N-acetals as N-acyliminium ion precursors: synthesis and absolute stereochemistry of epiquinamide.

A stereoselective synthesis of (+)-epiquinamide is presented in combination with determination of the absolute configuration of the natural product. Key steps in the sequence involved chemoenzymatic formation of an enantiomerically pure cyanohydrin, reductive cyclization to the corresponding cyclic N,N-acetal, and subsequent conversion into a suitable N-acyliminium ion precursor to enable construction of the second ring.

Behavioral characterization of the mGlu group II/III receptor antagonist, LY-341495, in animal models of anxiety and depression.

There is a growing body of evidence indicating that stimulation of metabotropic glutamate type II receptors (mGlu2/3) reduces anxiety in laboratory animals and humans. Surprisingly, it was reported that mGlu2/3 receptor antagonists have antidepressant- and anxiolytic-like activities in laboratory animal studies as well. The present study aimed to resolve this controversy by characterizing behavioral effects of a selective mGlu2/3 receptor antagonist, LY-341495, in a variety of animal models sensitive to clinically used anxiolytic and antidepressant agents. In agreement with previous reports, LY-341495 (0.3-3 mg/kg, i.p.) reduced immobility in the mouse forced swim test. LY-341495 was also effective in the marble burying test in mice, although similar effects were observed after administration of various drugs including methamphetamine. Further, LY-341495 had no effects in the elevated plus maze and stress-induced hyperthermia tests in mice, as well as on punished drinking (Geller-Seifter's test) and differential reinforcement of low rates of responding (DRL) in rats. It is concluded that behavioral profile of mGlu2/3 receptor antagonists as represented by LY-341495 is different from that of conventional anxiolytic and antidepressant drugs.

Amyloid beta oligomers (A beta(1-42) globulomer) suppress spontaneous synaptic activity by inhibition of P/Q-type calcium currents.

Abnormal accumulation of soluble oligomers of amyloid beta (Abeta) is believed to cause malfunctioning of neurons in Alzheimer's disease. It has been shown that Abeta oligomers impair synaptic plasticity, thereby altering the ability of the neuron to store information. We examined the underlying cellular mechanism of Abeta oligomer-induced synaptic modifications by using a recently described stable oligomeric Abeta preparation called "Abeta(1-42) globulomer." Synthetically prepared Abeta(1-42) globulomer has been shown to localize to neurons and impairs long-term potentiation (Barghorn et al., 2005). Here, we demonstrate that Abeta(1-42) globulomer does not affect intrinsic neuronal properties, as assessed by measuring input resistance and discharge characteristics, excluding an unspecific alteration of membrane properties. We provide evidence that Abeta(1-42) globulomer, at concentrations as low as 8 nM, specifically suppresses spontaneous synaptic activity resulting from a reduction of vesicular release at terminals of both GABAergic and glutamatergic synapses. EPSCs and IPSCs were primarily unaffected. A detailed search for the precise molecular target of Abeta(1-42) globulomer revealed a specific inhibition of presynaptic P/Q calcium currents, whereas other voltage-activated calcium currents remained unaltered. Because intact P/Q calcium currents are needed for synaptic plasticity, the disruption of such currents by Abeta(1-42) globulomer may cause deficits in cellular mechanisms of information storage in brains of Alzheimer's disease patients. The inhibitory effect of Abeta(1-42) globulomer on synaptic vesicle release could be reversed by roscovitine, a specific enhancer of P/Q currents. Selective enhancement of the P/Q calcium current may provide a promising strategy in the treatment of Alzheimer's disease.

Biocatalytic reductions: from lab curiosity to "first choice".

Enzyme-catalyzed reductions have been studied for decades and have been introduced in more than 10 industrial processes for production of various chiral alcohols, alpha-hydroxy acids and alpha-amino acids. The earlier hurdle of expensive cofactors was taken by the development of highly efficient cofactor regeneration methods. In addition, the accessible number of suitable dehydrogenases and therefore the versatility of this technology is constantly increasing and currently expanding beyond asymmetric production of alcohols and amino acids. Access to a large set of enzymes for highly selective C=C reductions and reductive amination of ketones for production of chiral secondary amines and the development of improved D-selective amino acid dehydrogenases will fuel the next wave of industrial bioreduction processes.

Laser capture microdissection and microarray analysis of dividing neural progenitor cells from the adult rat hippocampus.

Neural progenitor cells reside in the hippocampus of adult rodents and humans and generate granule neurons throughout life. Knowledge about the molecular processes regulating these neurogenic cells is fragmentary. In order to identify genes with a role in the proliferation of adult neural progenitor cells, a protocol was elaborated to enable the staining and isolation of such cells under RNA-preserving conditions with a combination of immunohistochemistry and laser capture microdissection. We increased proliferation of neural progenitor cells by electroconvulsive treatment, one of the most effective antidepressant treatments, and isolated Ki-67-positive cells using this new protocol. RNA amplification via in vitro transcription and subsequent microarray analysis revealed over 100 genes that were differentially expressed in neural progenitor cells due to electroconvulsive treatment compared to untreated control animals. Some of these genes have already been implicated in the functioning of neural progenitor cells or have been induced by electroconvulsive treatment; these include brain-derived neurotrophic factor (Bdnf), PDZ-binding kinase (Pbk) and abnormal spindle-like microcephaly-associated (Aspm). In addition, genes were identified for which no role in the proliferation of neurogenic progenitors has been described so far, such as enhancer of zeste homolog 2 (Ezh2).