OV329, a novel highly potent γ-aminobutyric acid aminotransferase inactivator, induces pronounced anticonvulsant effects in the pentylenetetrazole seizure threshold test and in amygdala-kindled rats.

OBJECTIVE: An attractive target to interfere with epileptic brain hyperexcitability is the enhancement of γ-aminobutyric acidergic (GABAergic) inhibition by inactivation of the GABA-metabolizing enzyme GABA aminotransferase (GABA-AT). GABA-AT inactivators were designed to control seizures by raising brain GABA levels. OV329, a novel drug candidate for the treatment of epilepsy and addiction, has been shown in vitro to be substantially more potent as a GABA-AT inactivator than vigabatrin, an antiseizure drug approved as an add-on therapy for adult patients with refractory complex partial seizures and monotherapy for pediatric patients with infantile spasms. Thus, we hypothesized that OV329 should produce pronounced anticonvulsant effects in two different rat seizure models. METHODS: We therefore examined the effects of OV329 (5, 20, and 40 mg/kg ip) on the seizure threshold of female Wistar Unilever rats, using the timed intravenous pentylenetetrazole (ivPTZ) seizure threshold model as a seizure test particularly sensitive to GABA-potentiating manipulations, and amygdala-kindled rats as a model of difficult-to-treat temporal lobe epilepsy. RESULTS: GABA-AT inactivation by OV329 clearly increased the threshold of both ivPTZ-induced and amygdala-kindled seizures. OV329 further showed a 30-fold greater anticonvulsant potency on ivPTZ-induced myoclonic jerks and clonic seizures compared to vigabatrin investigated previously. Notably, all rats were responsive to OV329 in both seizure models. SIGNIFICANCE: These results reveal an anticonvulsant profile of OV329 that appears to be superior in both potency and efficacy to vigabatrin and highlight OV329 as a highly promising candidate for the treatment of seizures and pharmacoresistant epilepsies.

Soticlestat, a novel cholesterol 24-hydroxylase inhibitor, reduces seizures and premature death in Dravet syndrome mice.

OBJECTIVE: Dravet syndrome is a severe developmental and epileptic encephalopathy (DEE) most often caused by de novo pathogenic variants in SCN1A. Individuals with Dravet syndrome rarely achieve seizure control and have significantly elevated risk for sudden unexplained death in epilepsy (SUDEP). Heterozygous deletion of Scn1a in mice (Scn1a+/- ) recapitulates several core phenotypes, including temperature-dependent and spontaneous seizures, SUDEP, and behavioral abnormalities. Furthermore, Scn1a+/- mice exhibit a similar clinical response to standard anticonvulsants. Cholesterol 24-hydroxlase (CH24H) is a brain-specific enzyme responsible for cholesterol catabolism. Recent research has indicated the therapeutic potential of CH24H inhibition for diseases associated with neural excitation, including seizures. METHODS: In this study, the novel compound soticlestat, a CH24H inhibitor, was administered to Scn1a+/- mice to investigate its ability to improve Dravet-like phenotypes in this preclinical model. RESULTS: Soticlestat treatment reduced seizure burden, protected against hyperthermia-induced seizures, and completely prevented SUDEP in Scn1a+/- mice. Video-electroencephalography (EEG) analysis confirmed the ability of soticlestat to reduce occurrence of electroclinical seizures. SIGNIFICANCE: This study demonstrates that soticlestat-mediated inhibition of CH24H provides therapeutic benefit for the treatment of Dravet syndrome in mice and has the potential for treatment of DEEs.

The STARS Phase 2 Study: A Randomized Controlled Trial of Gaboxadol in Angelman Syndrome.

OBJECTIVE: To evaluate safety and tolerability and exploratory efficacy end points for gaboxadol (OV101) compared with placebo in individuals with Angelman syndrome (AS). METHODS: Gaboxadol is a highly selective orthosteric agonist that activates δ-subunit-containing extrasynaptic γ-aminobutyric acid type A (GABAA) receptors. In a multicenter, double-blind, placebo-controlled, parallel-group trial, adolescent and adult individuals with a molecular diagnosis of AS were randomized (1:1:1) to 1 of 3 dosing regimens for a duration of 12 weeks: placebo morning dose and gaboxadol 15 mg evening dose (qd), gaboxadol 10 mg morning dose and 15 mg evening dose (bid), or placebo morning and evening dose. Safety and tolerability were monitored throughout the study. Prespecified exploratory efficacy end points included adapted Clinical Global Impression-Severity and Clinical Global Impression-Improvement (CGI-I) scales, which documented the clinical severity at baseline and change after treatment, respectively. RESULTS: Eighty-eight individuals were randomized. Of 87 individuals (aged 13-45 years) who received at least 1 dose of study drug, 78 (90%) completed the study. Most adverse events (AEs) were mild to moderate, and no life-threatening AEs were reported. Efficacy of gaboxadol, as measured by CGI-I improvement in an exploratory analysis, was observed in gaboxadol qd vs placebo (p = 0.0006). CONCLUSION: After 12 weeks of treatment, gaboxadol was found to be generally well-tolerated with a favorable safety profile. The efficacy as measured by the AS-adapted CGI-I scale warrants further studies. CLINICALTRIALSGOV IDENTIFIER: NCT02996305. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that, for individuals with AS, gaboxadol is generally safe and well-tolerated.

Platelet-Reactive Antibodies in Patients after Ischaemic Stroke-An Epiphenomenon or a Natural Protective Mechanism.

Ischaemic brain damage induces autoimmune responses, including the production of autoantibodies with potential neuroprotective effects. Platelets share unexplained similarities with neurons, and the formation of anti-platelet antibodies has been documented in neurological disorders. The aim of this study was to investigate the presence of anti-platelet antibodies in the peripheral blood of patients after ischaemic stroke and determine any clinical correlations. Using a flow cytometry-based platelet immunofluorescence method, we detected platelet-reactive antibodies in 15 of 48 (31%) stroke patients and two of 50 (4%) controls (p < 0.001). Western blotting revealed heterogeneous reactivities with platelet proteins, some of which overlapped with brain proteins. Stroke patients who carried anti-platelet antibodies presented with larger infarcts and more severe neurological dysfunction, which manifested as higher scores on the National Institutes of Health Stroke Scale (NIHSS; p = 0.009), but they had a greater recovery in the NIHSS by the time of hospital discharge (day 7 ± 2) compared with antibody-negative patients (p = 0.043). Antibodies from stroke sera reacted more strongly with activated platelets (p = 0.031) and inhibited platelet aggregation by up to 30.1 ± 2.8% (p < 0.001), suggesting the potential to interfere with thrombus formation. In conclusion, platelet-reactive antibodies can be found in patients soon after ischaemic stroke and correlate with better short-term outcomes, suggesting a potential novel mechanism limiting thrombosis.

Soticlestat, a novel cholesterol 24-hydroxylase inhibitor shows a therapeutic potential for neural hyperexcitation in mice.

Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol, the primary mechanism of cholesterol catabolism in the brain. The therapeutic potential of CH24H activation has been extensively investigated, whereas the effects of CH24H inhibition remain poorly characterized. In this study, the therapeutic potential of CH24H inhibition was investigated using a newly identified small molecule, soticlestat (TAK-935/OV935). The biodistribution and target engagement of soticlestat was assessed in mice. CH24H-knockout mice showed a substantially lower level of soticlestat distribution in the brain than wild-type controls. Furthermore, brain-slice autoradiography studies demonstrated the absence of [3H]soticlestat staining in CH24H-knockout mice compared with wild-type mice, indicating a specificity of soticlestat binding to CH24H. The pharmacodynamic effects of soticlestat were characterized in a transgenic mouse model carrying mutated human amyloid precursor protein and presenilin 1 (APP/PS1-Tg). These mice, with excitatory/inhibitory imbalance and short life-span, yielded a remarkable survival benefit when bred with CH24H-knockout animals. Soticlestat lowered brain 24S-hydroxycholesterol in a dose-dependent manner and substantially reduced premature deaths of APP/PS1-Tg mice at a dose lowering brain 24S-hydroxycholesterol by approximately 50%. Furthermore, microdialysis experiments showed that soticlestat can suppress potassium-evoked extracellular glutamate elevations in the hippocampus. Taken together, these data suggest that soticlestat-mediated inhibition of CH24H may have therapeutic potential for diseases associated with neural hyperexcitation.

Gaboxadol Normalizes Behavioral Abnormalities in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and autism. FXS is also accompanied by attention problems, hyperactivity, anxiety, aggression, poor sleep, repetitive behaviors, and self-injury. Recent work supports the role of γ-aminobutyric-acid (GABA), the primary inhibitory neurotransmitter in the brain, in mediating symptoms of FXS. Deficits in GABA machinery have been observed in a mouse model of FXS, including a loss of tonic inhibition in the amygdala, which is mediated by extrasynaptic GABAA receptors. Humans with FXS also show reduced GABAA receptor availability. Here, we sought to evaluate the potential of gaboxadol (also called OV101 and THIP), a selective and potent agonist for delta-subunit-containing extrasynaptic GABAA receptors (dSEGA), as a therapeutic agent for FXS by assessing its ability to normalize aberrant behaviors in a relatively uncharacterized mouse model of FXS (Fmr1 KO2 mice). Four behavioral domains (hyperactivity, anxiety, aggression, and repetitive behaviors) were probed using a battery of behavioral assays. The results showed that Fmr1 KO2 mice were hyperactive, had abnormal anxiety-like behavior, were more irritable and aggressive, and had an increased frequency of repetitive behaviors compared to wild-type (WT) littermates, which are all behavioral deficits reminiscent of individuals with FXS. Treatment with gaboxadol normalized all of the aberrant behaviors observed in Fmr1 KO2 mice back to WT levels, providing evidence of its potential benefit for treating FXS. We show that the potentiation of extrasynaptic GABA receptors alone, by gaboxadol, is sufficient to normalize numerous behavioral deficits in the FXS model using endpoints that are directly translatable to the clinical presentation of FXS. Taken together, these data support the future evaluation of gaboxadol in individuals with FXS, particularly with regard to symptoms of hyperactivity, anxiety, irritability, aggression, and repetitive behaviors.

Olanzapine: A potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics.

Designer receptors exclusively activated by designer drugs (DREADDs) derived from muscarinic receptors not only are a powerful tool to test causality in basic neuroscience but also are potentially amenable to clinical translation. A major obstacle, however, is that the widely used agonist clozapine N-oxide undergoes conversion to clozapine, which penetrates the blood-brain barrier but has an unfavorable side effect profile. Perlapine has been reported to activate DREADDs at nanomolar concentrations but is not approved for use in humans by the Food and Drug Administration or the European Medicines Agency, limiting its translational potential. Here, we report that the atypical antipsychotic drug olanzapine, widely available in various formulations, is a potent agonist of the human M4 muscarinic receptor-based DREADD, facilitating clinical translation of chemogenetics to treat central nervous system diseases.

Gene therapy reduces Parkinson's disease symptoms by reorganizing functional brain connectivity.

Gene therapy is emerging as a promising approach for treating neurological disorders, including Parkinson's disease (PD). A phase 2 clinical trial showed that delivering glutamic acid decarboxylase (GAD) into the subthalamic nucleus (STN) of patients with PD had therapeutic effects. To determine the mechanism underlying this response, we analyzed metabolic imaging data from patients who received gene therapy and those randomized to sham surgery, all of whom had been scanned preoperatively and at 6 and 12 months after surgery. Those who received GAD gene therapy developed a unique treatment-dependent polysynaptic brain circuit that we termed as the GAD-related pattern (GADRP), which reflected the formation of new polysynaptic functional pathways linking the STN to motor cortical regions. Patients in both the treatment group and the sham group expressed the previously reported placebo network (the sham surgery-related pattern or SSRP) when blinded to the treatment received. However, only the appearance of the GADRP correlated with clinical improvement in the gene therapy-treated subjects. Treatment-induced brain circuits can thus be useful in clinical trials for isolating true treatment responses and providing insight into their underlying biological mechanisms.

N-methyl-d-aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines.

BACKGROUND: N-methyl-d-aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro- and anti-differentiating effects have been shown in different contexts. OBJECTIVES: The aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells. METHODS: Meg-01, Set-2, and K-562 leukemic cell lines were differentiated using phorbol-12-myristate-13-acetate (PMA, 10 nmol L-1) or valproic acid (VPA, 500 µmol L-1). Normal megakaryocytes were grown from mouse marrow-derived hematopoietic progenitors (lineage-negative and CD41a-enriched) in the presence of thrombopoietin (30-40 nmol L-1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK-801 (100 µmol L-1); their effects compared against appropriate controls. RESULTS: The most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK-801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg-01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves. CONCLUSIONS: NMDAR-mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

A clinical-grade gene therapy vector for pharmacoresistant epilepsy successfully overexpresses NPY in a human neuronal cell line.

PURPOSE: Epilepsy is a common neurological condition characterised by recurrent unprovoked seizures and often treatable with appropriate medication. However, almost 30% of cases are pharmacoresistant and while a proportion of these may be amenable to resective surgery, a gene therapy approach could be an attractive alternative option. Neuropeptide Y (NPY) has anticonvulsant and anti-epileptogenic properties in animal models of temporal lobe epilepsy when delivered by an adeno-associated viral (AAV) vector. Here we sought to demonstrate successful secretion of NPY from AAV-transduced human neuronal cells, which would be essential in planning any clinical trial. METHODS: A human neuroblastoma cell line (SH-SY5Y) was used to assess in vitro whether an AAV vector manufactured to clinical-grade protocols would be effective at transducing these cells to express NPY. Optimal transduction efficiency was first achieved with retinoic acid and tetradecanoylphorpol-13-acetate (TPA) treatment, prior to expose to AAV1-green fluorescent protein (GFP) reporter vector, AAV1-NPY therapeutic vector or sham treated with no vector. Levels of NPY in cell supernatants were determined using two antibody-based methods RESULTS: We found that the levels of NPY released into the cell culture media supernatant, and protein extracts of the cell pellet, were significantly higher following exposure to AAV1-NPY than when compared to either a control GFP reporter vector (AAV1-GFP) or sham treated controls. CONCLUSION: This first demonstration that an AAV-NPY construct can successfully transduce human neuronal cells supports the pre-clinical development of a clinical trial using AAV-based NPY for pharmacoresistant epilepsy.

Long-term restoration of visual function in end-stage retinal degeneration using subretinal human melanopsin gene therapy.

Optogenetic strategies to restore vision in patients who are blind from end-stage retinal degenerations aim to render remaining retinal cells light sensitive once photoreceptors are lost. Here, we assessed long-term functional outcomes following subretinal delivery of the human melanopsin gene (OPN4) in the rd1 mouse model of retinal degeneration using an adeno-associated viral vector. Ectopic expression of OPN4 using a ubiquitous promoter resulted in cellular depolarization and ganglion cell action potential firing. Restoration of the pupil light reflex, behavioral light avoidance, and the ability to perform a task requiring basic image recognition were restored up to 13 mo following injection. These data suggest that melanopsin gene therapy via a subretinal route may be a viable and stable therapeutic option for the treatment of end-stage retinal degeneration in humans.

Cdc25A Is a Critical Mediator of Ischemic Neuronal Death In Vitro and In Vivo.

Dysregulation of cell cycle machinery is implicated in a number of neuronal death contexts, including stroke. Increasing evidence suggests that cyclin-dependent kinases (Cdks) are inappropriately activated in mature neurons under ischemic stress conditions. We previously demonstrated a functional role for the cyclin D1/Cdk4/pRb (retinoblastoma tumor suppressor protein) pathway in delayed neuronal death induced by ischemia. However, the molecular signals leading to cyclin D/Cdk4/pRb activation following ischemic insult are presently not clear. Here, we investigate the cell division cycle 25 (Cdc25) dual-specificity phosphatases as potential upstream regulators of ischemic neuronal death and Cdk4 activation. We show that a pharmacologic inhibitor of Cdc25 family members (A, B, and C) protects mouse primary neurons from hypoxia-induced delayed death. The major contributor to the death process appears to be Cdc25A. shRNA-mediated knockdown of Cdc25A protects neurons in a delayed model of hypoxia-induced death in vitro Similar results were observed in vivo following global ischemia in the rat. In contrast, neurons singly or doubly deficient for Cdc25B/C were not significantly protective. We show that Cdc25A activity, but not level, is upregulated in vitro following hypoxia and global ischemic insult in vivo Finally, we show that shRNA targeting Cdc25A blocks Ser795 pRb phosphorylation. Overall, our results indicate a role for Cdc25A in delayed neuronal death mediated by ischemia.SIGNIFICANCE STATEMENT A major challenge in stroke is finding an effective neuroprotective strategy to treat cerebral ischemic injury. Cdc25 family member A (Cdc25A) is a phosphatase normally activated during cell division in proliferating cells. We found that Cdc25A is activated in neurons undergoing ischemic stress mediated by hypoxia in vitro and global cerebral ischemia in rats in vivo We show that pharmacologic or genetic inhibition of Cdc25A activity protects neurons from delayed death in vitro and in vivo Downregulation of Cdc25A led to reduction in retinoblastoma tumor suppressor protein (pRb) phosphorylation. An increase in pRb phosphorylation has been previously linked to ischemic neuronal death. Our results identify Cdc25A as a potential target for neuroprotectant strategy for the treatment of delayed ischemic neuronal death.

Long-term follow-up of a randomized AAV2-GAD gene therapy trial for Parkinson's disease.

BACKGROUND. We report the 12-month clinical and imaging data on the effects of bilateral delivery of the glutamic acid decarboxylase gene into the subthalamic nuclei (STN) of advanced Parkinson's disease (PD) patients. METHODS. 45 PD patients were enrolled in a 6-month double-blind randomized trial of bilateral AAV2-GAD delivery into the STN compared with sham surgery and were followed for 12 months in open-label fashion. Subjects were assessed with clinical outcome measures and 18F-fluorodeoxyglucose (FDG) PET imaging. RESULTS. Improvements under the blind in Unified Parkinson's Disease Rating Scale (UPDRS) motor scores in the AAV2-GAD group compared with the sham group continued at 12 months [time effect: F(4,138) = 11.55, P < 0.001; group effect: F(1,35) = 5.45, P < 0.03; repeated-measures ANOVA (RMANOVA)]. Daily duration of levodopa-induced dyskinesias significantly declined at 12 months in the AAV2-GAD group (P = 0.03; post-hoc Bonferroni test), while the sham group was unchanged. Analysis of all FDG PET images over 12 months revealed significant metabolic declines (P < 0.001; statistical parametric mapping RMANOVA) in the thalamus, striatum, and prefrontal, anterior cingulate, and orbitofrontal cortices in the AAV2-GAD group compared with the sham group. Across all time points, changes in regional metabolism differed for the two groups in all areas, with significant declines only in the AAV2-GAD group (P < 0.005; post-hoc Bonferroni tests). Furthermore, baseline metabolism in the prefrontal cortex (PFC) correlated with changes in motor UPDRS scores; the higher the baseline PFC metabolism, the better the clinical outcome. CONCLUSION. These findings show that clinical benefits after gene therapy with STN AAV2-GAD in PD patients persist at 12 months. TRIAL REGISTRATION. ClinicalTrials.gov NCT00643890. FUNDING. Neurologix Inc.

Inhibition of NMDA receptor function with an anti-GluN1-S2 antibody impairs human platelet function and thrombosis.

GluN1 is a mandatory component of N-methyl-D-aspartate receptors (NMDARs) best known for their roles in the brain, but with increasing evidence for relevance in peripheral tissues, including platelets. Certain anti-GluN1 antibodies reduce brain infarcts in rodent models of ischaemic stroke. There is also evidence that human anti-GluN1 autoantibodies reduce neuronal damage in stroke patients, but the underlying mechanism is unclear. This study investigated whether anti-GluN1-mediated neuroprotection involves inhibition of platelet function. Four commercial anti-GluN1 antibodies were screened for their abilities to inhibit human platelet aggregation. Haematological parameters were examined in rats vaccinated with GluN1. Platelet effects of a mouse monoclonal antibody targeting the glycine-binding region of GluN1 (GluN1-S2) were tested in assays of platelet activation, aggregation and thrombus formation. The epitope of anti-GluN1-S2 was mapped and the mechanism of antibody action modelled using crystal structures of GluN1. Our work found that rats vaccinated with GluN1 had a mildly prolonged bleeding time and carried antibodies targeting mostly GluN1-S2. The monoclonal anti-GluN1-S2 antibody (from BD Biosciences) inhibited activation and aggregation of human platelets in the presence of adrenaline, adenosine diphosphate, collagen, thrombin and a protease-activated receptor 1-activating peptide. When human blood was flowed over collagen-coated surfaces, anti-GluN1-S2 impaired thrombus growth and stability. The epitope of anti-GluN1-S2 was mapped to α-helix H located within the glycine-binding clamshell of GluN1, where the antibody binding was computationally predicted to impair opening of the NMDAR channel. Our results indicate that anti-GluN1-S2 inhibits function of human platelets, including dense granule release and thrombus growth. Findings add to the evidence that platelet NMDARs regulate thrombus formation and suggest a novel mechanism by which anti-GluN1 autoantibodies limit stroke-induced neuronal damage.

Selected GRIN2A mutations in melanoma cause oncogenic effects that can be modulated by extracellular glutamate.

GRIN2A mutations are frequent in melanoma tumours but their role in disease is not well understood. GRIN2A encodes a modulatory subunit of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that certain GRIN2A mutations increase NMDAR function and support melanoma growth through oncogenic effects. This hypothesis was tested using 19 low-passage melanoma cell lines, four of which carried novel missense mutations in GRIN2A that we previously reported. We examined NMDAR expression, function of a calcium ion (Ca2+) channel and its contribution to cell growth using pharmacological modulators; findings were correlated with the presence or absence of GRIN2A mutations. We found that NMDAR expression was low in all melanoma cell lines, independent of GRIN2A mutations. In keeping with this, NMDAR-mediated Ca2+ influx and its contribution to cell proliferation were weak in most cell lines. However, certain GRIN2A mutations and culture media with lower glutamate levels enhanced NMDAR effects on cell growth and invasion. The main finding was that G762E was associated with higher glutamate-mediated Ca2+ influx and stronger NMDAR contribution to cell proliferation, compared with wild-type GRIN2A and other GRIN2A mutations. The pro-invasive phenotype of mutated cell lines was increased in culture medium containing less glutamate, implying environmental modulation of mutation effects. In conclusion, NMDAR ion channel function is low in cultured melanoma cells but supports cell proliferation and invasion. Selected GRIN2A mutations, such as G762E, are associated with oncogenic consequences that can be modulated by extracellular glutamate. Primary cultures may be better suited to determine the role of the NMDAR in melanoma in vivo.

A Therapeutic Role for Survivin in Mitigating the Harmful Effects of Ionizing Radiation.

Background. Radiation therapy is a form of adjuvant care used in many oncological treatment protocols. However, nonmalignant neighboring tissues are harmed as a result of this treatment. Therefore, the goal of this study was to induce the production of survivin, an antiapoptotic protein, to determine if this protein could provide protection to noncancerous cells during radiation exposure. Methods. Using a murine model, a recombinant adenoassociated virus (rAAV) was used to deliver survivin to the treatment group and yellow fluorescence protein (YFP) to the control group. Both groups received targeted radiation. Visual inspection, gait analysis, and tissue histology were used to determine the extent of damage caused by the radiation. Results. The YFP group demonstrated ulceration of the irradiated area while the survivin treated mice exhibited only hair loss. Histology showed that the YFP treated mice experienced dermal thickening, as well as an increase in collagen that was not present in the survivin treated mice. Gait analysis demonstrated a difference between the two groups, with the YFP mice averaging a lower speed. Conclusions. The use of gene-modification to induce survivin expression in normal tissues allows for the protection of nontarget areas from the negative side effects normally associated with ionizing radiation.

Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation and supports differentiation.

Human megakaryocytes release glutamate and express glutamate-gated Ca(2+)-permeable N-methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca(2+) responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca(2+) flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca(2+) entry into Meg-01 cells, amplified Ca(2+) responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5-100µM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100µM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC50 20µM and 36µM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation. In conclusion, NMDARs provide a novel pathway for Ca(2+) entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts.

Adipose VEGF Links the White-to-Brown Fat Switch With Environmental, Genetic, and Pharmacological Stimuli in Male Mice.

Living in an enriched environment (EE) decreases adiposity, increases energy expenditure, causes resistance to diet induced obesity, and induces brown-like (beige) cells in white fat via activating a hypothalamic-adipocyte axis. Here we report that EE stimulated vascular endothelial growth factor (VEGF) expression in a fat depot-specific manner prior to the emergence of beige cells. The VEGF up-regulation was independent of hypoxia but required intact sympathetic tone to the adipose tissue. Targeted adipose overexpression of VEGF reproduced the browning effect of EE. Adipose-specific VEGF knockout or pharmacological VEGF blockade with antibodies abolished the induction of beige cell by EE. Hypothalamic brain-derived neurotrophic factor stimulated by EE regulated the adipose VEGF expression, and VEGF signaling was essential to the hypothalamic brain-derived neurotrophic factor-induced white adipose tissue browning. Furthermore, VEGF signaling was essential to the beige cells induction by exercise, a ß3-adrenergic agonist, and a peroxisome proliferator-activated receptor-γ ligand, suggesting a common downstream pathway integrating diverse upstream mechanisms. Exploiting this pathway may offer potential therapeutic interventions to obesity and metabolic diseases.

Social overcrowding as a chronic stress model that increases adiposity in mice.

Stress is a widely recognized risk factor for psychiatric and metabolic disorders. A number of animal models utilizing various stressors have been developed to facilitate our understanding in the pathophysiology of stress-related dysfunctions. The most commonly used chronic stress paradigms include the unpredictable chronic mild stress paradigm, the social defeat paradigm and the social deprivation paradigm. Here we assess the potential of social crowding as an alternative chronic stress model to study the effects on affective behaviors and metabolic disturbances. Ten-week-old male C57BL/6 mice were housed in groups of four (control) or eight (social crowding; SC) in standard cage for 9 weeks. Exploration, anxiety- and depressive-like behaviors were assessed in the open field test, the elevated T-maze, the novelty-suppressed feeding test and the forced swim test. SC mice exhibited a modest anxiety-like phenotype without change in depressive-like behaviors. Nine weeks of social crowding did not affect the body weight, but robustly increased adiposity as determined by increased mass of fat depots. Consistent with the increased fat content, serum leptin was markedly elevated in the SC mice. Specific changes in gene expression were also observed in the hypothalamus and the white adipose tissue following SC housing. Our study demonstrates the potential of social crowding as an alternative model for the study of stress-related metabolic and behavioral dysfunctions.

Network modulation following sham surgery in Parkinson's disease.

Patient responses to placebo and sham effects are a major obstacle to the development of therapies for brain disorders, including Parkinson's disease (PD). Here, we used functional brain imaging and network analysis to study the circuitry underlying placebo effects in PD subjects randomized to sham surgery as part of a double-blind gene therapy trial. Metabolic imaging was performed prior to randomization, then again at 6 and 12 months after sham surgery. In this cohort, the sham response was associated with the expression of a distinct cerebello-limbic circuit. The expression of this network increased consistently in patients blinded to treatment and correlated with independent clinical ratings. Once patients were unblinded, network expression declined toward baseline levels. Analogous network alterations were not seen with open-label levodopa treatment or during disease progression. Furthermore, sham outcomes in blinded patients correlated with baseline network expression, suggesting the potential use of this quantitative measure to identify "sham-susceptible" subjects before randomization. Indeed, Monte Carlo simulations revealed that a priori exclusion of such individuals substantially lowers the number of randomized participants needed to demonstrate treatment efficacy. Individualized subject selection based on a predetermined network criterion may therefore limit the need for sham interventions in future clinical trials.