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... more
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 model...
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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... more
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 (18)F-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 g...
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... more
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 (Ca(2+)) 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 Ca(2+) 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 NMDA...
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Background: Widespread application of gene therapy has been limited by several factors with one of the most important being that systemic gene therapy methods do not allow for specific targeting of tissues. Methods aimed at improving this... more
Background: Widespread application of gene therapy has been limited by several factors with one of the most important being that systemic gene therapy methods do not allow for specific targeting of tissues. Methods aimed at improving this specificity are needed to achieve clinically applicable gene therapy treatment regimens. Recent reports have described the delivery of genes into muscle flaps for local production of gene products. These studies have relied on time consuming methods that use ex vivo perfusion of ...
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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... more
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 pres...
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Abstract: The present invention relates to viral vectors and in particular to adeno-associated viral vectors with enhanced gene transfer capabilities into cells of the central nervous system (CNS). The invention has been developed... more
Abstract: The present invention relates to viral vectors and in particular to adeno-associated viral vectors with enhanced gene transfer capabilities into cells of the central nervous system (CNS). The invention has been developed primarily for gene transfer in glial cells and further for the treatment of diseases associated with glial cell pathology.
Recombinant AAV is increasingly becoming the vector of choice for many gene therapy applications in the CNS, due to its lack of toxicity and high level of sustained expression. With recent improvements in the generation of pure, high... more
Recombinant AAV is increasingly becoming the vector of choice for many gene therapy applications in the CNS, due to its lack of toxicity and high level of sustained expression. With recent improvements in the generation of pure, high titer vector stocks, the regulation of gene expression is now a key issue for successful translation of gene therapy-based treatments to the clinic. The level of the transgene protein may need to be maintained within a narrow therapeutic window for the successful treatment of human disease. The doxycycline responsive system directs a dose-responsive, tightly regulated level of gene expression and has been used successfully in transgenic mouse models. Here, we have optimized an autoregulatory, bidirectional doxycyline responsive cassette specifically for use in rAAV. We minimized the size of the cassette and decreased the basal leakiness of the system, leading to tight regulation in the rat
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Chronic central administration of NPY has dramatic effects on energy balance, however, the exact role of the hypothalamic paraventricular nucleus (PVN) in this is unknown. The aim of this study was to further unravel the contribution of... more
Chronic central administration of NPY has dramatic effects on energy balance, however, the exact role of the hypothalamic paraventricular nucleus (PVN) in this is unknown. The aim of this study was to further unravel the contribution of NPY signaling in the PVN to energy balance. Recombinant adeno-associated viral particles containing NPY (rAAV-NPY) were injected in the rat brain with coordinates targeted at the PVN. For three weeks, body weight, food intake, endocrine parameters, body temperature and locomotor activity were measured. Furthermore, effects on insulin sensitivity and expression of NPY, AgRP and POMC in the arcuate nucleus were studied. Food intake was increased specifically in the light period. Furthermore, dark phase body temperature and locomotor activity were reduced. This resulted in obesity characterized by increased fat mass, elevated plasma insulin, leptin and adiponectin, decreased AgRP expression in the arcuate nucleus and decreased insulin sensitivity, where...
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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... more
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 VEGF expression in a fat depot-specific manner prior to the emergence of beige cells. The VEGF upregulation 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 BDNF stimulated by EE regulated the adipose VEGF expression and VEGF signaling was essential to the hypothalamic BDNF-induced WAT browning. Furthermore, VEGF signaling was essential to the beige cells induction by exercise, a β3 adrenergic agonist, and a PPARγ ligand, suggesting a common downstream pathway integrating div...
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The authors investigated the association between socioeconomic position and stage of breast cancer at the time of diagnosis in a nationwide Danish study. All 28 765 women with a primary invasive breast cancer diagnosed between 1983 and... more
The authors investigated the association between socioeconomic position and stage of breast cancer at the time of diagnosis in a nationwide Danish study. All 28 765 women with a primary invasive breast cancer diagnosed between 1983 and 1999 were identified in a nationwide clinical database and information on socioeconomic variables was obtained from Statistics Denmark. The risk of being diagnosed with a high-risk breast cancer, that is size >20 mm, lymph-node positive, ductal histology/high histologic grade and hormone receptor negative, was analysed by multivariate logistic regression. The adjusted odds ratio (OR) for high-risk breast cancer was reduced with longer education with a 12% reduced risk (95% confidence interval (CI), 0.80,0.96) in women with higher education and increased with reduced disposable income (low income group: OR, 1.22; 95% CI, 1.10,1.34). There was an urban-rural gradient, with higher risk among rural women (OR 1.10; 95 % CI, 1.02, 1.18) and lower risk am...
Research Interests: Higher Education, Breast Cancer, British, Apoptosis, Logistic Regression, and 20 moreNature, Cell Division, Adipose tissue, Denmark, Postmenopausal Women, Humans, Female, Incidence, Aged, Middle Aged, Premenopausal women, Rural Women, Adult, Odds ratio, Cancer cells, Low Income, Lymph Node, Cancers, Confidence Interval, and Socioeconomic Factors
An incomplete understanding of the pathological processes involved in neurodegeneration and dysfunction of spinal cord injuries and diseases makes these disorders difficult to treat. Repair of damaged or genetically impaired spinal cord... more
An incomplete understanding of the pathological processes involved in neurodegeneration and dysfunction of spinal cord injuries and diseases makes these disorders difficult to treat. Repair of damaged or genetically impaired spinal cord also has been limited by the complexity, cellular heterogeneity, and relative inaccessibility of the tissue. Thus, therapeutic options for the treatment of either chronic spinal cord diseases such as amyotrophic lateral sclerosis or acute spinal cord injuries have been rather limited. Potential new therapeutic targets are being identified as our understanding of the molecular pathology involved in neural injury and regeneration increases. Recent advances in gene transfer techniques have made gene therapy a more realistic and viable strategy for the treatment of a broad range of spinal cord disorders. This review summarizes the current state of knowledge regarding the limitations and recent advances in gene therapy and potential application of this te...
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Inappropriate activation of cell cycle proteins, in particular cyclin D/Cdk4, is implicated in neuronal death induced by various pathologic stresses, including DNA damage and ischemia. Key targets of Cdk4 in proliferating cells include... more
Inappropriate activation of cell cycle proteins, in particular cyclin D/Cdk4, is implicated in neuronal death induced by various pathologic stresses, including DNA damage and ischemia. Key targets of Cdk4 in proliferating cells include members of the E2F transcription factors, which mediate the expression of cell cycle proteins as well as death-inducing genes. However, the presence of multiple E2F family members complicates our understanding of their role in death. We focused on whether E2F4, an E2F member believed to exhibit crucial control over the maintenance of a differentiated state of neurons, may be critical in ischemic neuronal death. We observed that, in contrast to E2F1 and E2F3, which sensitize to death, E2F4 plays a crucial protective role in neuronal death evoked by DNA damage, hypoxia, and global ischemic insult both in vitro and in vivo. E2F4 occupies promoter regions of proapoptotic factors, such as B-Myb, under basal conditions. Following stress exposure, E2F4-p130 ...
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In Parkinson's disease, long-term dopamine replacement therapy is complicated by the appearance of L-DOPA-induced dyskinesia (LID). One major hypothesis is that LID results from an aberrant transcriptional program in striatal neurons... more
In Parkinson's disease, long-term dopamine replacement therapy is complicated by the appearance of L-DOPA-induced dyskinesia (LID). One major hypothesis is that LID results from an aberrant transcriptional program in striatal neurons induced by L-DOPA and triggered by the activation of ERK. To identify these genes, we performed transcriptome analyses in the striatum in 6-hydroxydopamine-lesioned mice. A time course analysis (0-6 h after treatment with L-DOPA) identified an acute signature of 709 genes, among which genes involved in protein phosphatase activity were overrepresented, suggesting a negative feedback on ERK activation by l-DOPA. l-DOPA-dependent deregulation of 28 genes was blocked by pretreatment with SL327, an inhibitor of ERK activation, and 26 genes were found differentially expressed between highly and weakly dyskinetic animals after treatment with L-DOPA. The intersection list identified five genes: FosB, Th, Nptx2, Nedd4l, and Ccrn4l. Nptx2 encodes neuronal pe...
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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... more
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 cyclin D1/Cdk4/pRb pathway in delayed neuronal death induced by ischemia. However, the molecular signal(s) leading to cyclin D/Cdk4/pRb activation following ischemic insult is 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 & 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...
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... more
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, sug...
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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... more
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 crowd...
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ABSTRACT Most chemotherapy regimens rely on systemic administration of drugs leading to a wide array of toxicities. Using viral-vector-mediated gene modification of muscle tissues, we have developed a method for gene-directed enzyme... more
ABSTRACT Most chemotherapy regimens rely on systemic administration of drugs leading to a wide array of toxicities. Using viral-vector-mediated gene modification of muscle tissues, we have developed a method for gene-directed enzyme prodrug therapy that allows for localized drug administration. An inactive prodrug of geldanamycin was activated locally for inhibition of tumor growth without systemic toxicities. A recombinant adeno-associated virus (rAAV) was used to deliver β-galactosidase (LacZ) to the treatment group and green fluorescent protein to the control group. After 1 week, both groups received adenocarcinoma cells in the same location as the previous rAAV injection. The geldanamycin prodrug was administered 1 h later via intraperitoneal injection. Tumor growth was significantly suppressed in animals whose muscles were gene modified to express β-galactosidase compared with the control. Serum assay to access hepatotoxicity resulted in no significant differences between the animals treated with the inactive or activated form of geldanamycin, indicating minimal damage to non-target organs. Using gene-directed enzyme prodrug therapy, in combination with novel recombinant AAV vectors, we have developed a method for localized activation of chemotherapeutic agents that limits the toxicities seen with traditional systemic administration of these potent drugs.Cancer Gene Therapy advance online publication, 19 September 2014; doi:10.1038/cgt.2014.47.
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A major focus of biology is determining the role that specific genes play within a cell. The ability to control gene expression is a powerful tool for biologists, and methods that facilitate the expression of genes in trans have been very... more
A major focus of biology is determining the role that specific genes play within a cell. The ability to control gene expression is a powerful tool for biologists, and methods that facilitate the expression of genes in trans have been very useful in delineating gene function. However, methods that enable researchers to decrease or inhibit gene expression in a controlled and specific manner have been lacking. A significant advancement in the field of gene silencing is the recent discovery of RNA interference (RNAi), a post-transcriptional gene silencing mechanism first described in C. elegans and Drosophila and more recently shown to occur in mammals, including mouse and human cells. This chapter will examine several aspects of RNAi and its related gene silencing mechanisms and will discuss the impact that technologies based on these endogenous cellular processes have had on studies of gene function and on new approaches to the treatment of disease.
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The delivery of exogenous genes into the brain is becoming an increasingly important strategy for answering questions about the molecular mechanisms of brain function. Answers to these questions may be applied to many of the disorders... more
The delivery of exogenous genes into the brain is becoming an increasingly important strategy for answering questions about the molecular mechanisms of brain function. Answers to these questions may be applied to many of the disorders that affect the brain. For example, a detailed understanding of the mechanisms that modulate long-term changes in neurotransmitter release will almost certainly lead to new approaches to diseases such as the epilepsies, in which neurotransmitter release is altered. Knowledge of the molecular means by which neurotransmitters shape neuronal development and cause neurodegeneration, or how trophic factors regulate neuronal health, will lead to insights into how defects in these pathways cause specific diseases. To answer these questions, we have developed a defective herpes simplex virus (HSV) system for the delivery of exogenous genes into the brain. This system directs precise spatial and temporal expression of recombinant genes in the brain. We discuss ...
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Adipose tissue plays an essential role in metabolic homeostasis, and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here we... more
Adipose tissue plays an essential role in metabolic homeostasis, and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant AAV vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass, morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue.
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Research Interests: Stroke, Humans, Mice, Female, Animals, and 11 moreMale, Brain Ischemia, Neurons, Clinical Sciences, Aged, Middle Aged, Rats, Adult, Biological markers, Neurosciences, and Autoantibodies
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Abstract Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene transfer into the central nervous system (CNS). However, a major hurdle for gene delivery to the mammalian brain is to achieve high-level... more
Abstract Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene transfer into the central nervous system (CNS). However, a major hurdle for gene delivery to the mammalian brain is to achieve high-level transduction in target cells beyond the immediate injection site. Therefore, in addition to improvements in expression cassettes and viral titers, optimal injection parameters need to be defined. Here, we show that previous studies of somatic cell gene transfer to the mammalian brain have used suboptimal ...
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... Parkinson's Disease. Michael G. Kaplitt 1,|[ast]| , David Eidelberg 2 , Andrew Feigin 2 , Helen Fitzimmons 3,|[ast]| , Patricia Lawlor 4 , Kristin Strybing 1 , Marie Cox 2 , ChrisTang 2 , Ross Bland 3,|[ast]| and Matthew J.... more
... Parkinson's Disease. Michael G. Kaplitt 1,|[ast]| , David Eidelberg 2 , Andrew Feigin 2 , Helen Fitzimmons 3,|[ast]| , Patricia Lawlor 4 , Kristin Strybing 1 , Marie Cox 2 , ChrisTang 2 , Ross Bland 3,|[ast]| and Matthew J. During 1,|[ast]|. ...