Knockdown of NEAT1 prevents post-stroke lipid droplet agglomeration in microglia by regulating autophagy.

BACKGROUND: Lipid droplets (LD), lipid-storing organelles containing neutral lipids like glycerolipids and cholesterol, are increasingly accepted as hallmarks of inflammation. The nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA with over 200 nucleotides, exerts an indispensable impact on regulating both LD agglomeration and autophagy in multiple neurological disorders. However, knowledge as to how NEAT1 modulates the formation of LD and associated signaling pathways is limited. METHODS: In this study, primary microglia were isolated from newborn mice and exposed to oxygen-glucose-deprivation/reoxygenation (OGD/R). To further explore NEAT1-dependent mechanisms, an antisense oligonucleotide (ASO) was adopted to silence NEAT1 under in vitro conditions. Studying NEAT1-dependent interactions with regard to autophagy and LD agglomeration under hypoxic conditions, the inhibitor and activator of autophagy 3-methyladenine (3-MA) and rapamycin (RAPA) were used, respectively. In a preclinical stroke model, mice received intraventricular injections of ASO NEAT1 or control vectors in order to yield NEAT1 knockdown. Analysis of readout parameters included qRT-PCR, immunofluorescence, western blot assays, and behavioral tests. RESULTS: Microglia exposed to OGD/R displayed a temporal pattern of NEAT1 expression, peaking at four hours of hypoxia followed by six hours of reoxygenation. After effectively silencing NEAT1, LD formation and autophagy-related proteins were significantly repressed in hypoxic microglia. Stimulating autophagy in ASO NEAT1 microglia under OGD/R conditions by means of RAPA reversed the downregulation of LD agglomeration and perilipin 2 (PLIN2) expression. On the contrary, application of 3-MA promoted repression of both LD agglomeration and expression of the LD-associated protein PLIN2. Under in vivo conditions, NEAT1 was significantly increased in mice at 24 h post-stroke. Knockdown of NEAT1 significantly alleviated LD agglomeration and inhibited autophagy, resulting in improved cerebral perfusion, reduced brain injury and increased neurological recovery. CONCLUSION: NEAT1 is a key player of LD agglomeration and autophagy stimulation, and NEAT1 knockdown provides a promising therapeutic value against stroke.

TREM2 regulates microglial lipid droplet formation and represses post-ischemic brain injury.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor protein predominantly expressed in microglia within the central nervous system (CNS). TREM2 regulates multiple microglial functions, including lipid metabolism, immune reaction, inflammation, and microglial phagocytosis. Recent studies have found that TREM2 is highly expressed in activated microglia after ischemic stroke. However, the role of TREM2 in the pathologic response after stroke remains unclear. Herein, TREM2-deficient microglia exhibit an impaired phagocytosis rate and cholesteryl ester (CE) accumulation, leading to lipid droplet formation and upregulation of Perilipin-2 (PLIN2) expression after hypoxia. Knockdown of TREM2 results in increased lipid synthesis (PLIN2, SOAT1) and decreased cholesterol clearance and lipid hydrolysis (LIPA, ApoE, ABCA1, NECH1, and NPC2), further impacting microglial phenotypes. In these lipid droplet-rich microglia, the TGF-ß1/Smad2/3 signaling pathway is downregulated, driving microglia towards a pro-inflammatory phenotype. Meanwhile, in a neuron-microglia co-culture system under hypoxic conditions, we found that microglia lost their protective effect against neuronal injury and apoptosis when TREM2 was knocked down. Under in vivo conditions, TREM2 knockdown mice express lower TGF-ß1 expression levels and a lower number of anti-inflammatory M2 phenotype microglia, resulting in increased cerebral infarct size, exacerbated neuronal apoptosis, and aggravated neuronal impairment. Our work suggests that TREM2 attenuates stroke-induced neuroinflammation by modulating the TGF-ß1/Smad2/3 signaling pathway. TREM2 may play a direct role in the regulation of inflammation and also exert an influence on the post-ischemic inflammation and the stroke pathology progression via regulation of lipid metabolism processes. Thus, underscoring the therapeutic potential of TREM2 agonists in ischemic stroke and making TREM2 an attractive new clinical target for the treatment of ischemic stroke and other inflammation-related diseases.

TGF-ß1 Decreases Microglia-Mediated Neuroinflammation and Lipid Droplet Accumulation in an In Vitro Stroke Model.

Hypoxia triggers reactive microglial inflammation and lipid droplet (LD) accumulation under stroke conditions, although the mutual interactions between these two processes are insufficiently understood. Hence, the involvement of transforming growth factor (TGF)-ß1 in inflammation and LD accumulation in cultured microglia exposed to hypoxia were analyzed herein. Primary microglia were exposed to oxygen-glucose deprivation (OGD) injury and lipopolysaccharide (LPS) stimulation. For analyzing the role of TGF-ß1 patterns under such conditions, a TGF-ß1 siRNA and an exogenous recombinant TGF-ß1 protein were employed. Further studies applied Triacsin C, an inhibitor of LD formation, in order to directly assess the impact of LD formation on the modulation of inflammation. To assess mutual microglia-to-neuron interactions, a co-culture model of these cells was established. Upon OGD exposure, microglial TGF-ß1 levels were significantly increased, whereas LPS stimulation yielded decreased levels. Elevating TGF-ß1 expression proved highly effective in suppressing inflammation and reducing LD accumulation in microglia exposed to LPS. Conversely, inhibition of TGF-ß1 led to the promotion of microglial cell inflammation and an increase in LD accumulation in microglia exposed to OGD. Employing the LD formation inhibitor Triacsin C, in turn, polarized microglia towards an anti-inflammatory phenotype. Such modulation of both microglial TGF-ß1 and LD levels significantly affected the resistance of co-cultured neurons. This study provides novel insights by demonstrating that TGF-ß1 plays a protective role against microglia-mediated neuroinflammation through the suppression of LD accumulation. These findings offer a fresh perspective on stroke treatment, suggesting the potential of targeting this pathway for therapeutic interventions.

Preconditioned extracellular vesicles from hypoxic microglia reduce poststroke AQP4 depolarization, disturbed cerebrospinal fluid flow, astrogliosis, and neuroinflammation.

Background: Stroke stimulates reactive astrogliosis, aquaporin 4 (AQP4) depolarization and neuroinflammation. Preconditioned extracellular vesicles (EVs) from microglia exposed to hypoxia, in turn, reduce poststroke brain injury. Nevertheless, the underlying mechanisms of such effects are elusive, especially with regards to inflammation, AQP4 polarization, and cerebrospinal fluid (CSF) flow. Methods: Primary microglia and astrocytes were exposed to oxygen-glucose deprivation (OGD) injury. For analyzing the role of AQP4 expression patterns under hypoxic conditions, a co-culture model of astrocytes and microglia was established. Further studies applied a stroke model, where some mice also received an intracisternal tracer infusion of rhodamine B. As such, these in vivo studies involved the analysis of AQP4 polarization, CSF flow, astrogliosis, and neuroinflammation as well as ischemia-induced brain injury. Results: Preconditioned EVs decreased periinfarct AQP4 depolarization, brain edema, astrogliosis, and inflammation in stroke mice. Likewise, EVs promoted postischemic CSF flow and cerebral blood perfusion, and neurological recovery. Under in vitro conditions, hypoxia stimulated M2 microglia polarization, whereas EVs augmented M2 microglia polarization and repressed M1 microglia polarization even further. In line with this, astrocytes displayed upregulated AQP4 clustering and proinflammatory cytokine levels when exposed to OGD, which was reversed by preconditioned EVs. Reduced AQP4 depolarization due to EVs, however, was not a consequence of unspecific inflammatory regulation, since LPS-induced inflammation in co-culture models of astrocytes and microglia did not result in altered AQP4 expression patterns in astrocytes. Conclusions: These findings show that hypoxic microglia may participate in protecting against stroke-induced brain damage by regulating poststroke inflammation, astrogliosis, AQP4 depolarization, and CSF flow due to EV release.

Recycling nitrogen from liquid digestate via novel reactive struvite and zeolite minerals to mitigate agricultural pollution.

Recycling nutrients is of paramount importance. For this reason, struvite and nitrogen enriched zeolite fertilizers produced from wastewater treatments are receiving growing attention in European markets. However, their effects on agricultural soils are far from certain, especially struvite, which only recently was implemented in EU Fertilizing Product Regulations. In this paper, we investigate the effects of these materials in acid sandy arable soil, particularly focusing on N dynamics, evaluating potential losses, transformation pathways, and the effects of struvite and zeolitic tuffs on main soil biogeochemical parameters, in comparison to traditional fertilization with digestate. Liming effect (pH alkalinization) was observed in all treatments with varying intensities, affecting most of the soil processes. The struvite was quickly solubilized due to soil acidity, and the release of nutrients stimulated nitrifying and denitrifying microorganisms. Zeolitic tuff amendments decreased the NOx gas emissions, which are precursors to the powerful climate altering N2O gas, and the N enriched chabazite tuff also recorded smaller NH3 emissions compared to the digestate. However, a high dosage of zeolites in soil increased NH3 emissions after fertilization, due to pronounced pH shifts. Contrasting effects were observed between the two zeolitic tuffs when applied as soil amendments; while the chabazite tuff had a strong positive effect - increasing up to ∼90% the soil microbial N immobilization - the employed clinoptilolite tuff had immediate negative effects on the microbial biomass, likely due to the large quantities of sulphur released. However, when applied at lower dosages, the N enriched clinoptilolite also contributed to the increase of microbial N. From these outcomes, we confirm the potential of struvite and zeolites to mitigate the outfluxes of nutrients from agricultural systems. To gain the best results and significantly lower environmental impacts, extension practitioners could give recommendations based on the soils that are planned for zeolite application.

Adult alcohol drinking and emotional tone are mediated by neutral sphingomyelinase during development in males.

Alcohol use, abuse, and addiction, and resulting health hazards are highly sex-dependent with unknown mechanisms. Previously, strong links between the SMPD3 gene and its coded protein neutral sphingomyelinase 2 (NSM) and alcohol abuse, emotional behavior, and bone defects were discovered and multiple mechanisms were identified for females. Here we report strong sex-dimorphisms for central, but not for peripheral mechanisms of NSM action in mouse models. Reduced NSM activity resulted in enhanced alcohol consumption in males, but delayed conditioned rewarding effects. It enhanced the acute dopamine response to alcohol, but decreased monoaminergic systems adaptations to chronic alcohol. Reduced NSM activity increased depression- and anxiety-like behavior, but was not involved in alcohol use for the self-management of the emotional state. Constitutively reduced NSM activity impaired structural development in the brain and enhanced lipidomic sensitivity to chronic alcohol. While the central effects were mostly opposite to NSM function in females, similar roles in bone-mediated osteocalcin release and its effects on alcohol drinking and emotional behavior were observed. These findings support the view that the NSM and multiple downstream mechanism may be a source of the sex-differences in alcohol use and emotional behavior.

Neutral sphingomyelinase mediates the co-morbidity trias of alcohol abuse, major depression and bone defects.

Mental disorders are highly comorbid and occur together with physical diseases, which are often considered to arise from separate pathogenic pathways. We observed in alcohol-dependent patients increased serum activity of neutral sphingomyelinase. A genetic association analysis in 456,693 volunteers found associations of haplotypes of SMPD3 coding for NSM-2 (NSM) with alcohol consumption, but also with affective state, and bone mineralisation. Functional analysis in mice showed that NSM controls alcohol consumption, affective behaviour, and their interaction by regulating hippocampal volume, cortical connectivity, and monoaminergic responses. Furthermore, NSM controlled bone-brain communication by enhancing osteocalcin signalling, which can independently supress alcohol consumption and reduce depressive behaviour. Altogether, we identified a single gene source for multiple pathways originating in the brain and bone, which interlink disorders of a mental-physical co-morbidity trias of alcohol abuse-depression/anxiety-bone disorder. Targeting NSM and osteocalcin signalling may, thus, provide a new systems approach in the treatment of a mental-physical co-morbidity trias.

The selective FKBP51 inhibitor SAFit2 reduces alcohol consumption and reinstatement of conditioned alcohol effects in mice.

There is still no widely effective pharmacotherapy for alcohol addiction available in the clinic. FK506-binding protein 51 (FKBP51) is a negative regulator of the glucocorticoid receptor signaling pathway that regulates the stress-induced glucocorticoid feedback circuit. Here we asked whether selective inhibitors of FKBP51, exemplified by SAFit2, may serve as a new pharmacological strategy to reduce alcohol consumption and conditioned alcohol effects in a mouse model. We report that a relatively short treatment with SAFit2 (20 mg/kg, ip) reduces ongoing 16 vol% alcohol consumption when administered during free access to alcohol in a two-bottle free-choice test. SAFit2 was also able to reduce alcohol consumption when given during an abstinence period immediately before relapse. In contrast, SAFit2 did not affect alcohol consumption when given during a relapse period after repeated withdrawal from alcohol. SAFit2 (10 and 20 mg/kg, ip) showed no effects when used in an intermittent drinking schedule. When 20 vol% alcohol was only available every other day, SAFit2 had no effect on drinking, no matter whether given during a drinking episode or the day before. SAFit2 (2 and 20 mg/kg, ip) did not affect the expression of an alcohol-induced conditioned place preference (CPP). However, SAFit2 was able to inhibit alcohol-induced reinstatement of an extinguished CPP in a dose-dependent way. Altogether, these data may suggest pharmacological inhibition of FKBP51 as a viable strategy to reduce alcohol seeking and consumption.

Ceramides affect alcohol consumption and depressive-like and anxiety-like behavior in a brain region- and ceramide species-specific way in male mice.

Depression and alcohol dependence are associated with increased plasma ceramide concentrations in humans. Pharmacological increase in C16 ceramide concentrations in the dorsal hippocampus (DH) induced a depressive-like phenotype in naïve mice. However, the effects of C16 ceramide on alcohol consumption and anxiety-like behavior as well as the behavioral effects of other ceramide species are yet unknown. Therefore, we investigated whether repeated infusion of ceramides with different fatty acid chain lengths (C8, C16, and C20) into the DH and the basolateral amygdala (BLA) alter alcohol consumption, emotional behavior, and tissue monoamine levels. Our results revealed that C16, but not C8 and C20, ceramide altered alcohol drinking and emotional behavior in a brain region-specific way without altering tissue noradrenaline, dopamine, and serotonin levels in the prefrontal cortex, ventral striatum, and dorsal mesencephalon. In more detail, C16 ceramide increased alcohol consumption when infused into the BLA, but not when infused into the DH. Furthermore, C16 ceramide induced a depressive-like phenotype when infused into the DH, but a predominantly anxiogenic-like phenotype (in a non-social, but not a social context) when infused into the BLA. In turn, alcohol drinking normalized C16 ceramide-induced depressive-like and anxiogenic-like phenotypes. This study demonstrates a complex ceramide species-specific and brain region-specific modulation of alcohol consumption and emotional behavior in mice and provides the framework for future studies investigating the involvement of distinct ceramide species in the regulation of emotional behavior.

Effects of conditioned social fear on ethanol drinking and vice-versa in male mice.

RATIONALE: Social anxiety disorder (SAD) is highly comorbid with alcohol use disorders, but the complex relationship between social fear and alcohol drinking is poorly understood due to the lack of specific animal models. OBJECTIVES: We investigated whether social fear alters ethanol drinking under both stress-free and stress-inducing conditions and whether ethanol alleviates symptoms of social fear. METHODS: We used the social fear conditioning (SFC) paradigm, an animal model with face and predictive validity to SAD, to induce specific social fear in male CD1 mice, i.e., without comorbid depression or anxiety-like behavior. Plasma corticosterone (CORT) levels were measured in conditioned (SFC+) and unconditioned (SFC-) mice after exposure to non-social or social stimuli. Ethanol drinking was assessed in the two-bottle free-choice paradigm (1) for 16 days under stress-free conditions and (2) for 6 h after exposure to social stimuli. The effects of ethanol drinking and social fear on anxiety-like behavior and taste preference were tested in the elevated plus-maze and sucrose and quinine preference tests. RESULTS: We show that exposure to social but not non-social stimuli leads to higher plasma CORT levels in SFC+ compared with SFC- mice. We also show that social fear decreases voluntary ethanol consumption under stress-free conditions, but increases ethanol consumption after exposure to social stimuli. Ethanol drinking, on the other hand, reduces social fear without altering anxiety-like behavior, locomotor activity, and taste preference. CONCLUSIONS: These results have important clinical connotations as they suggest that voluntary ethanol drinking might specifically reverse symptoms of social fear in a SAD-relevant animal model.

Prenatal androgen receptor activation determines adult alcohol and water drinking in a sex-specific way.

Alcohol use disorders are major psychiatric disorders. Correlational studies in humans suggested organizational hormonal effects during embryonic development as a risk factor for adult alcohol dependence. Permanent changes can be induced by the activity of sex hormones, like testosterone. Here, we demonstrate a relationship between prenatal androgen receptor (AR)-activation and adult alcohol as well as water drinking in mice in a sex-dependent fashion. Prenatal AR inhibition using the antagonist flutamide decreased adult male alcohol consumption. In contrast, prenatal AR activation by dihydrotestosterone (DHT) led to an increase in adult alcohol consumption in females. These effects were different in adult water drinking, flutamide increased water consumption in females and DHT increased water consumption in males. Prenatal flutamide reduced locomotion and anxiety in adult males but was ineffective in females. We found that prenatal AR activation controls adult levels of monoaminergic modulatory transmitters in the brain and blood hormone levels in a sex-specific way. RNA-Seq analysis confirmed a prenatal AR mediated control of adult expression of alcohol drinking-related genes like Bdnf and Per2. These findings demonstrate that prenatal androgen activity is a risk factor for the establishment of alcohol consumption in adults by its organizational effects.

Prenatal androgen-receptor activity has organizational morphological effects in mice.

Prenatal sex hormones exert organizational effects. It has been suggested that prenatal sex hormones affect adult morphological parameters, such as the finger length. Especially the second-to-fourth finger length (2D:4D) ratio has been implicated to be modified when exposed to higher androgen levels in utero. Here we show in a mouse model that experimental manipulation of the prenatal androgen level, by blocking the androgen receptor with flutamide or activating the androgen receptor with dihydrotestosterone (DHT), leads to changes in the length of the fingers of all paws in males and females. In addition to that, also total paw length and the 2D:4D ratio was affected. In males treated with DHT, the 2D:4D ratio was increased, while flutamide-treatment in females led to a reduced 2D:4D ratio. We also measured other parameters, such as head size, body length and tail length and demonstrate that body morphology is affected by prenatal androgen exposure with more prominent effects in females. Another factor that is thought to be influenced by early androgens is handedness. We tested mice for handedness, but did not find a significant effect of the prenatal treatment. These findings demonstrate that prenatal androgen activity is involved in the development of body morphology and might be a useful marker for prenatal androgen exposure.

Paradoxical antidepressant effects of alcohol are related to acid sphingomyelinase and its control of sphingolipid homeostasis.

Alcohol is a widely consumed drug that can lead to addiction and severe brain damage. However, alcohol is also used as self-medication for psychiatric problems, such as depression, frequently resulting in depression-alcoholism comorbidity. Here, we identify the first molecular mechanism for alcohol use with the goal to self-medicate and ameliorate the behavioral symptoms of a genetically induced innate depression. An induced over-expression of acid sphingomyelinase (ASM), as was observed in depressed patients, enhanced the consumption of alcohol in a mouse model of depression. ASM hyperactivity facilitates the establishment of the conditioned behavioral effects of alcohol, and thus drug memories. Opposite effects on drinking and alcohol reward learning were observed in animals with reduced ASM function. Importantly, free-choice alcohol drinking-but not forced alcohol exposure-reduces depression-like behavior selectively in depressed animals through the normalization of brain ASM activity. No such effects were observed in normal mice. ASM hyperactivity caused sphingolipid and subsequent monoamine transmitter hypo-activity in the brain. Free-choice alcohol drinking restores nucleus accumbens sphingolipid- and monoamine homeostasis selectively in depressed mice. A gene expression analysis suggested strong control of ASM on the expression of genes related to the regulation of pH, ion transmembrane transport, behavioral fear response, neuroprotection and neuropeptide signaling pathways. These findings suggest that the paradoxical antidepressant effects of alcohol in depressed organisms are mediated by ASM and its control of sphingolipid homeostasis. Both emerge as a new treatment target specifically for depression-induced alcoholism.

Activin Controls Ethanol Potentiation of Inhibitory Synaptic Transmission Through GABAA Receptors and Concomitant Behavioral Sedation.

Activin, a member of the transforming growth factor-ß family, exerts multiple functions in the nervous system. Originally identified as a neurotrophic and -protective agent, increasing evidence implicates activin also in the regulation of glutamatergic and GABAergic neurotransmission in brain regions associated with cognitive and affective functions. To explore how activin impacts on ethanol potentiation of GABA synapses and related behavioral paradigms, we used an established transgenic model of disrupted activin receptor signaling, in which mice express a dominant-negative activin receptor IB mutant (dnActRIB) under the control of the CaMKIIα promoter. Comparison of GABAA receptor currents in hippocampal neurons from dnActRIB mice and wild-type mice showed that all concentrations of ethanol tested (30-150 mM) produced much stronger potentiation of phasic inhibition in the mutant preparation. In dentate granule cells of dnActRIB mice, tonic GABA inhibition was more pronounced than in wild-type neurons, but remained insensitive to low ethanol (30 mM) in both preparations. The heightened ethanol sensitivity of phasic inhibition in mutant hippocampi resulted from both pre- and postsynaptic mechanisms, the latter probably involving PKCɛ. At the behavioral level, ethanol produced significantly stronger sedation in dnActRIB mice than in wild-type mice, but did not affect consumption of ethanol or escalation after withdrawal. We link the abnormal narcotic response of dnActRIB mice to ethanol to the excessive potentiation of inhibitory neurotransmission. Our study suggests that activin counteracts oversedation from ethanol by curtailing its augmenting effect at GABA synapses.

Outcome after Discontinuing Long-Term Benzimidazole Treatment in 11 Patients with Non-resectable Alveolar Echinococcosis with Negative FDG-PET/CT and Anti-EmII/3-10 Serology.

BACKGROUND/AIMS: Benzimidazoles are efficacious for treating non-resectable alveolar echinococcosis (AE), but their long-term parasitocidal (curative) effect is disputed. In this study, we prospectively analyzed the potential parasitocidal effect of benzimidazoles and whether normalization of FDG-PET/CT scans and anti-Emll/3-10-antibody levels could act as reliable "in vivo" parameters of AE-inactivation permitting to abrogate chemotherapy with a low risk for AE-recurrence. METHOD: This prospective study included 34 patients with non-resectable AE subdivided into group A (n = 11), followed-up after diagnosis and begin of chemotherapy at months 6, 12 and 24, and group B (n = 23) with a medium duration of chemotherapy of 10 (range 2-25) years. All patients were assessed by FDG-PET/CT examinations and anti-EmII/3-10 serology. Chemotherapy was abrogated in patients with normalization of FDG-PET/CT and serum anti-EmII/3-10 levels. These patients were closely followed-up for AE recurrence. Endpoint (parasitocidal efficacy) was defined by the absence of AE-recurrence >24 months after stopping treatment. RESULTS: Normalization of FDG-PET/CT scan and anti-EmII/3-10 levels occurred in 11 of 34 patients (32%). After abrogation of chemotherapy in these 11 patients, there was no evidence of AE-recurrence within a median of 70.5 (range 16-82) months. However, the patients' immunocompetence appears pivotal for the described long-term parasitocidal effect of benzimidazoles. CONCLUSIONS: The combination of negative FDG-PET/CT-scans and anti-EmII/3-10 antibody levels seem to be reliable parameters for assessing in vivo AE-larval inactivity after long-term benzimidazole chemotherapy. TRIAL REGISTRATION: clinicaltrials.gov: NCT00658294.

Serotonergic dysfunction in the A53T alpha-synuclein mouse model of Parkinson's disease.

Parkinson's disease, neuropathologically defined by the aggregation of α-synuclein, is characterized by neuropsychiatric symptoms such as depression and anxiety preceding the onset of motor symptoms. A loss of serotonergic neurons or their projections into the hippocampus and alterations in serotonin release may be linked to these symptoms. Here, we investigate the effect of human A53T α-synuclein on serotonergic neurons using 12-months-old transgenic mice. We detected human α-synuclein in the perikarya of brainstem median and dorsal raphe neurons as well as in serotonergic fibers in the hippocampus. Despite intracellular α-synuclein accumulation there was no loss of serotonergic neurons in dorsal and median raphe nuclei of A53T α-synuclein mice. However, serotonin levels were significantly reduced in the brainstem. In addition, serotonergic fiber density in the dorsal dentate gyrus was significantly less dense in transgenic mice. Interestingly, we detected a significantly compromised increase in doublecortin+ neuroblasts after chronic treatment with fluoxetine at the site of reduced serotonergic innervation, the infrapyramidal blade of the dorsal dentate gyrus in A53T α-synuclein mice. This suggests that α-synuclein affects serotonergic projections in a spatially distinct pattern within the hippocampus thereby influencing the response to antidepressant treatment.

Stirred batch crystallization of a therapeutic antibody fragment.

Technical-scale crystallization of therapeutic proteins may not only allow for a significant cost-reduction in downstream processing, but also enable new applications, e.g., the use of crystal suspensions for subcutaneous drug delivery. In this work, the crystallization of the antigen-binding fragment FabC225 was studied. First, vapor diffusion crystallization conditions from the literature were transferred to 10µL-scale microbatch experiments. A phase diagram was developed in order to identify the crystallization window. The conditions obtained from the microbatch experiments were subsequently transferred to parallelized 5mL-scale stirred-tank crystallizers. This scalable and reproducible agitated crystallization system allowed for an optimization of the crystallization process based on quantitative measurements. The optimized crystallization process resulted in an excellent yield of 99% in less than 2h by increasing the concentration of the crystallization agent ammonium sulfate during the process. The successful scalability of the Fab fragment crystallization process to 100mL-scale crystallizers based on geometric similarity was demonstrated. A favorable crystal size distribution was obtained. Furthermore, a wash step was introduced in order to remove unfavorable low-molecular substances from the crystals.

Pharmacological interference with metabotropic glutamate receptor subtype 7 but not subtype 5 differentially affects within- and between-session extinction of Pavlovian conditioned fear.

Fear extinction is defined as the attenuation of a conditioned-fear memory by re-exposing animals to the conditioned stimulus without the aversive stimulus. This process is known to be effectively enhanced via administration of D-cycloserine (DCS), a partial NMDA-receptor agonist. However, other glutamatergic mechanisms, such as interference with metabotropic glutamate receptor (mGluR) subtypes 5 and 7 in the extinction of aversive memories are insufficiently understood. Using the allosteric mGluR5 receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), the mGluR7 allosteric agonist N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082), and DCS for comparison, we aimed to study how pharmacological blockade of mGluR5 and activation of mGluR7 influenced within- and between-session conditioned-fear extinction training and extinction retention in rats. We show that when injected before extinction training, mGluR7 activation with AMN082 enhanced freezing and thereby attenuated within-session fear extinction, whereas both DCS and the mGluR5 receptor antagonist MPEP had no effect on this process. However, these differential drug effects were not long lasting, as no difference in extinction retention were observed 24 h later. Therefore, we assessed whether the compounds affect 24 h consolidation of extinction training following incomplete extinction training (between-session extinction). Similar to DCS, AMN082- but not MPEP-treated rats showed facilitated extinction retention, as exhibited by decreased freezing. Finally, using fluoxetine, we provide evidence that the effect of AMN082 on between-session extinction retention is most likely not via increasing 5-HT transmission. These findings demonstrate that mGluR7 activation differentially modulates conditioned-fear extinction, in dependence on the protocol employed, and suggests drugs with AMN082-like mechanisms as potential add-on drugs following exposure-based psychotherapy for fear-related human disorders.

Computer-assisted quantitative assessment of power Doppler US: effects of microbubble contrast agent in the differentiation of breast tumors.

RATIONALE AND OBJECTIVES: To objectively quantify the effects of a microbubble contrast agent to differentiate breast tumors with power doppler ultrasound and to compare these results with color doppler ultrasound (CD US). METHODS: In 47 patients a microbubble contrast agent was injected intravenously. Computer-assisted quantitative assessment of the color pixel density was performed to evaluate the increase in Doppler signals. Results were compared to previously published results of a color Doppler ultrasound study. RESULTS: Peak color pixel density at contrast-enhanced power Doppler ultrasound was higher for carcinomas than for benign tumors (P < 0.03). Time to peak enhancement was shorter in carcinomas than in benign tumors (P < 0.01). For both parameters, diagnostic accuracy of power Doppler ultrasound was 69 and 78%, and for color Doppler ultrasound 62 and 76%, respectively. CONCLUSIONS: Quantitative assessment of contrast-enhanced power Doppler ultrasound showed significant differences in malignant and benign breast tumors. Diagnostic accuracy of contrast-enhanced power Doppler ultrasound was higher compared to color Doppler ultrasound.

Benign breast lesions: minimally invasive vacuum-assisted biopsy with 11-gauge needles patient acceptance and effect on follow-up imaging findings.

PURPOSE: To evaluate patient acceptance of stereotactic or ultrasonographically (US) guided directional vacuum-assisted 11-gauge needle biopsy of breast lesions and short- and long-term changes at mammography and US resulting from the procedure. MATERIALS AND METHODS: For 91 benign lesions that had been sampled at either stereotactic or US-guided directional vacuum-assisted breast biopsy performed with 11-gauge needles, clinical, mammographic, and US changes were evaluated 1 week after biopsy; 6-month follow-up findings were available for 74 lesions. The subjective outcome of the procedure and patient satisfaction were assessed on the basis of a patient-completed questionnaire that incorporated a multistage scoring system. Statistical analysis of scores for condition for both biopsy methods was performed with the chi2 test. RESULTS: Adverse events occurred during the procedure in four patients. Clinically visible hematomas were observed at 1-week follow-up in 79% of patients. Densities were observed on mammograms in 46% of patients 1 week after biopsy; hematomas with a maximum diameter of 2 cm were seen on sonograms in 74%. Six months after biopsy, mammography revealed discrete architectural changes in one case. No abnormalities were found at 6-month follow-up US. Fifteen patients had various complaints during the procedure; six reported feeling constrained during the first few days after biopsy, and one patient was not satisfied with the cosmetic result. No patient reported a retrospective preference for surgical biopsy instead of directional vacuum-assisted biopsy. Analysis of scores for the stereotactic and US-guided methods revealed a significant difference (P <.001) in favor of the stereotactic method for condition during biopsy, while scores for condition in the first days after biopsy were more equally distributed between the two methods (P =.386). CONCLUSION: Directional vacuum-assisted 11-gauge needle biopsy of the breast is well accepted by patients and rarely produces changes that may alter the mammographic or sonographic appearance of the breast at 6-month follow-up.