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1.  PDE-4 Inhibition Rescues Aberrant Synaptic Plasticity in Drosophila and Mouse Models of Fragile X Syndrome 
The Journal of Neuroscience  2015;35(1):396-408.
Fragile X syndrome (FXS) is the leading cause of both intellectual disability and autism resulting from a single gene mutation. Previously, we characterized cognitive impairments and brain structural defects in a Drosophila model of FXS and demonstrated that these impairments were rescued by treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium. A well-documented biochemical defect observed in fly and mouse FXS models and FXS patients is low cAMP levels. cAMP levels can be regulated by mGluR signaling. Herein, we demonstrate PDE-4 inhibition as a therapeutic strategy to ameliorate memory impairments and brain structural defects in the Drosophila model of fragile X. Furthermore, we examine the effects of PDE-4 inhibition by pharmacologic treatment in the fragile X mouse model. We demonstrate that acute inhibition of PDE-4 by pharmacologic treatment in hippocampal slices rescues the enhanced mGluR-dependent LTD phenotype observed in FXS mice. Additionally, we find that chronic treatment of FXS model mice, in adulthood, also restores the level of mGluR-dependent LTD to that observed in wild-type animals. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of FXS is an important advance, in that this identifies and validates PDE-4 inhibition as potential therapeutic intervention for the treatment of individuals afflicted with FXS.
PMCID: PMC4287155  PMID: 25568131
cAMP; Drosophila; fragile X; memory; mouse; phosphodiesterase 4
2.  Noninvasive Imaging of Myocyte Apoptosis Following Application of a Stem Cell–Engineered Delivery Platform to Acutely Infarcted Myocardium 
The cardioprotective effects of mesenchymal stem cells (MSCs) include reducing myocyte apoptosis, and this effect can be enhanced by preconditioning and encapsulation in a fibrin scaffold. This study aimed to test the hypothesis that apoptosis imaging can detect the cardioprotective effects of a conditioned MSC patch grafted in a rat model of acute myocardial infarction.
Cell culture experiments simulating engraftment of fibrin patches onto beating rat ventricular myocytes exposed to hypoxia showed an effect of conditioned cells to reduce apoptosis. Twenty-three nude rats underwent successful left anterior descending coronary artery occlusion and were divided into 3 groups: transforming growth factor β1–conditioned human MSC-laden patches (CP), infarct alone without patch (no patch [NP]), and patch alone (patch only [PO]). Twenty-four hours after myocardial infarction, all rats were injected with 99mTc-hydrazinonicotinamide (99mTc-HYNIC) annexin V and 201Tl and underwent dual-isotope SPECT/CT imaging. Six rats were sacrificed for histology and counting. The remaining rats (n = 17; 1 rat was eliminated) were injected and imaged on day 7; of those, 3 rats were sacrificed for histology and counting, and the remaining 13 rats survived to day 21, when they were sacrificed for histology. Numbers of rats imaged on day 7 in the 3 groups were 7 in the CP group, 5 in the NP, and 5 in the PO. Perfused myocardium, infarct size, and 99mTc-HYNIC annexin V uptake were quantified from the scans from days 1 and 7. 99mTc-HYNIC annexin V uptake was correlated with quantitative caspase staining, and infarct size as percentage fibrosis was quantified at day 21.
99mTc-HYNIC annexin V uptake as percentage injected dose (×10−4) decreased between days 1 and 7 by 1.04 ± 0.28 in the CP group, 0.44 ± 0.17 in the NP group, and 0.34 ± 0.27 in the PO group (P = 0.003 for NP vs. CP, P = 0.005 for PO vs. CP, and P = 0.5 for NP vs. CP). The changes in defect size as percentage myocardium between days 1 and 7 were −8.83 ± 4.40 in the CP group, +1.00 ± 2.24 in the NP group, and −0.50 ± 4.20 in the PO group (P = 0.003 for NP vs. CP, P = 0.005 for PO vs. CP, and P = 0.50 for NP vs. PO). 99mTc-HYNIC annexin V uptake as percentage left ventricle by scanning correlated with caspase staining (r = 0.931, P = 0.002).
Transforming growth factor β1–conditioned human MSC-laden patches reduce myocyte apoptosis in the setting of acute infarction, and this effect can be detected by in vivo imaging with 99mTc-HYNIC annexin V.
PMCID: PMC4479399  PMID: 23616583
apoptosis; myocardial infarction; stem cells; radionuclides; engineered cardiac tissue
3.  Imaging RAGE expression in atherosclerotic plaques in hyperlipidemic pigs 
EJNMMI Research  2014;4:26.
Receptor for advanced glycated end product (RAGE) expression is a prominent feature of atherosclerosis. We have previously shown in apoE null mice uptake of a radiolabeled anti-RAGE antibody in atherosclerotic plaque and now evaluate RAGE-directed imaging to identify advanced plaques in a large animal model.
Nine hyperlipidemic (HL) pigs were injected with 603.1 ± 129.5 MBq of 99mTc-anti-RAGE F(ab′)2, and after 6 h (blood pool clearance), they underwent single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of the neck, thorax, and hind limbs. Two HL pigs received 99mTc non-immune IgG F(ab′)2, and three farm pigs were injected with 99mTc-anti-RAGE F(ab′)2. After imaging, the pigs were euthanized. The aorta from the root to bifurcation was dissected, and the innominates, proximal carotids, and coronaries were dissected and counted, stained for H&E and RAGE, and AHA-classified.
On pathology, 24% of the arterial segments showed AHA class III or IV lesions, and these lesions were confined almost exclusively to coronaries and carotids with % stenosis from 15% to 65%. Scatter plots of %ID/g for class III/IV vs. I/II lesions showed almost complete separation. Focal vascular uptake of tracer visualized on SPECT scans corresponded to class III/IV lesions in the coronary and carotid vessels. In addition, uptake in the hind limbs was noted in the HL pigs and corresponded to RAGE staining of small arteries in the muscle sections. Correlations for the vascular lesions were r = 0.747, P = 0.001 for %ID vs. %ID/g and r = 0.83, P = 0.002 for %ID/g vs. % RAGE staining.
Uptake of radiolabeled anti-RAGE antibody in coronary and carotid fibroatheroma and in the small arteries of the hind limbs in a relevant large animal model of atherosclerosis supports the important role of RAGE in atherosclerosis and peripheral artery disease as a target for imaging and treatment.
PMCID: PMC4078320  PMID: 25006545
RAGE; Atherosclerosis; Hyperlipidemic pigs; Imaging
4.  Beneficial Effect of Glucose Control on Atherosclerosis Progression in Diabetic ApoE−/− Mice: Shown by Rage Directed Imaging 
Objective. Receptor for advanced glycated endproducts (RAGE) plays an important role in atherogenesis in diabetes. We imaged RAGE to investigate the effect of glucose control to suppress RAGE and reduce atherosclerosis in apolipoprotein E null (apoE−/−) diabetic mice. Methods and Results. Thirty-three apoE−/− mice received streptozotocin and 6 weeks later 15 began treatment with insulin implants. Blood glucose measurements during study averaged: 140 ± 23 mg/dL (treated) and 354 ± 14 mg/dL (untreated). After 15 wk 30 mice were injected with 99mTc-anti-RAGE F(ab′)2, 3 with 99mTc-nonimmune IgG F(ab′)2, and all with CT contrast agent and underwent SPECT/CT imaging. At necropsy, the proximal aorta was weighed, counted, and sectioned and the % injected dose per gram (%ID/g) was calculated. From the merged SPECT/CT scans, tracer uptake localized to arteries was lower in the treated mice: 3.15 ± 1.82 × 10−3 versus 8.69 ± 4.58 × 10−3%ID (P = 0.001). Percent cross-sectional lesion area was smaller in the treated (14.3 ± 7.8% versus 29.5 ± 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Lesion size as percent cross-sectional area was smaller in the treated (14.3 ± 7.8% versus 29.5 ± 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Conclusions. These results support the importance of suppressing RAGE to reduce atherosclerotic complications of diabetes and value of molecular imaging to assess treatment effect.
PMCID: PMC4009322  PMID: 24829796
5.  The Drosophila DmGluRA is required for social interaction and memory 
Metabotropic glutamate receptors (mGluRs) have well-established roles in cognition and social behavior in mammals. Whether or not these roles have been conserved throughout evolution from invertebrate species is less clear. Mammals have eight mGluRs whereas Drosophila has a single DmGluRA, which has both Gi and Gq coupled signaling activity. We have utilized Drosophila to examine the role of DmGluRA in social behavior and various phases of memory. We have found that flies that are homozygous or heterozygous for loss of function mutations of DmGluRA have impaired social behavior in male Drosophila. Futhermore, flies that are heterozygous for loss of function mutations of DmGluRA have impaired learning during training, immediate-recall memory, short-term memory, and long-term memory as young adults. This work demonstrates a role for mGluR activity in both social behavior and memory in Drosophila.
PMCID: PMC3662090  PMID: 23720628
mGluR; memory; Drosophila; long-term memory; DmGluRA; learning
6.  Imaging receptor for advanced glycation end product expression in mouse model of hind limb ischemia 
EJNMMI Research  2013;3:37.
The purpose of this study is to image the effect of diabetes on expression of receptor for advanced glycation endproducts (RAGE) in limb ischemia in live animals.
Male wild-type C57BL/6 mice were either made diabetic or left as control. Two months later, diabetic and non-diabetic mice underwent left femoral artery ligation. The right leg served as lesion control. Five days later, mice were injected with 15.1 ± 4.4 MBq 99mTc-anti-RAGE F(ab’)2 and 4 to 5 h later (blood pool clearance) underwent SPECT/CT imaging. At the completion of imaging, mice were euthanized, hind limbs counted and sectioned, and scans reconstructed. Regions of interest were drawn on serial transverse sections comprising the hind limbs and activity in millicuries summed and divided by the injected dose (ID). Quantitative histology was performed for RAGE staining and angiogenesis.
Uptake of 99mTc-anti-RAGE F(ab')2 as %ID × 10−3 was higher in the left (ischemic) limbs for the diabetic mice (n = 8) compared to non-diabetic mice (n = 8) (1.20 ± 0.44% vs. 0.49 ± 0.40%; P = 0.0007) and corresponded to less angiogenesis in the diabetic mice. Uptake was also higher in the right limbs of diabetic compared to non-diabetic animals (0.82 ± 0.33% vs. 0.40 ± 0.14%; P = 0.0004).
These data show the feasibility of imaging and quantifying the effect of diabetes on RAGE expression in limb ischemia.
PMCID: PMC3662588  PMID: 23663412
Limb ischemia; Diabetes; RAGE; Molecular imaging; Radionuclides
7.  Pharmacological reversal of synaptic plasticity deficits in the mouse model of Fragile X syndrome by group II mGluR antagonist or lithium treatment 
Brain research  2010;1380:106-119.
Fragile X syndrome is the leading single gene cause of intellectual disabilities. Treatment of a Drosophila model of Fragile X syndrome with metabotropic glutamate receptor (mGluR) antagonists or lithium rescues social and cognitive impairments. A hallmark feature of the Fragile X mouse model is enhanced mGluR-dependent long-term depression (LTD) at Schaffer collateral to CA1 pyramidal synapses of the hippocampus. Here we examine the effects of chronic treatment of Fragile X mice in vivo with lithium or a group II mGluR antagonist on mGluR-LTD at CA1 synapses. We find that long term lithium treatment initiated during development (5–6 weeks of age) and continued throughout the lifetime of the Fragile X mice until 9–11 months of age restores normal mGluR-LTD. Additionally, chronic short term treatment beginning in adult Fragile X mice (8 weeks of age) with either lithium or an mGluR antagonist is also able to restore normal mGluR-LTD. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of Fragile X syndrome is an important advance, in that this identifies and validates these targets as potential therapeutic interventions for the treatment of individuals afflicted with Fragile X syndrome.
PMCID: PMC3050427  PMID: 21078304
Fragile X; FMRP; long-term depression (LTD); group II mGluR; lithium; Fragile X syndrome; metabotropic glutamate receptor; LY341495
8.  IgG3 deficiency extends lifespan and attenuates progression of glomerulonephritis in MRL/lpr mice 
Biology Direct  2012;7:3.
Antibodies of the IgG3 subclass have been implicated in the pathogenesis of the spontaneous glomerulonephritis observed in mice of the MRL/MpJ-Tnfrsf6lpr (MRL/lpr) inbred strain which have been widely studied as a model of systemic lupus erythematosus We have produced IgG3-deficient (-/-) mice with the MRL/lpr genetic background to determine whether IgG3 antibodies are necessary for or at least contributory to MRL/lpr-associated nephritis.
The gamma3 genotype (+/+ vs. +/- vs. -/-) did not appear to significantly affect serum titers of IgG auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin. However, while substantial serum titers of IgG3 auto-antibodies specific for double-stranded DNA (dsDNA) or α-actinin were seen in gamma3 +/+ mice, somewhat lower serum titers of these IgG3 auto-antibodies were found in gamma3 +/- mice, and gamma3 -/- mice exhibited baseline concentrations of these auto-antibodies. Analysis of immunoglobulins eluted from snap-frozen kidneys obtained from mice of all three gamma3 genotypes at ~18 weeks of age revealed much higher quantities of IgG in the kidneys from gamma3 +/+ than gamma3 -/- mice, and most IgG eluted from +/+ mice was IgG3. The serum creatinine levels in gamma3 +/+ mice substantially exceeded those of age-matched gamma3 -/- mice after ~21 weeks of age. Histopathological examination of kidneys from mice sacrificed at pre-determined ages also revealed more extensive glomerulosclerosis in gamma3 +/+ or +/- mice than in -/- mice beginning at 21 weeks of age. Survival analysis for IgG3-deficient and IgG3-producing MRL/lpr mice revealed that gamma3 -/- mice lived significantly longer (p = 0.0006) than either gamma3 +/- or +/+ mice. Spontaneous death appeared to be due to irreversible renal failure, because > 85% of glomeruli in kidneys from mice that died spontaneously were obliterated by glomerulosclerosis.
The available evidence suggests that IgG3 deficiency partially protects MRL/lpr mice against glomerulonephritis-associated morbidity and mortality by slowing or arresting the progression to glomerulosclerosis.
This article was reviewed by Pushpa Pandiyan, Irun Cohen, and Etienne Joly.
PMCID: PMC3293080  PMID: 22248284
9.  Age-dependent cognitive impairment in a Drosophila Fragile X model and its pharmacological rescue 
Biogerontology  2009;11(3):347-362.
Fragile X syndrome afflicts 1 in 2,500 individuals and is the leading heritable cause of mental retardation worldwide. The overriding clinical manifestation of this disease is mild to severe cognitive impairment. Age-dependent cognitive decline has been identified in Fragile X patients, although it has not been fully characterized nor examined in animal models. A Drosophila model of this disease has been shown to display phenotypes bearing similarity to Fragile X symptoms. Most notably, we previously identified naive courtship and memory deficits in young adults with this model that appear to be due to enhanced metabotropic glutamate receptor (mGluR) signaling. Herein we have examined age-related cognitive decline in the Drosophila Fragile X model and found an age-dependent loss of learning during training. We demonstrate that treatment with mGluR antagonists or lithium can prevent this age-dependent cognitive impairment. We also show that treatment with mGluR antagonists or lithium during development alone displays differential efficacy in its ability to rescue naive courtship, learning during training and memory in aged flies. Furthermore, we show that continuous treatment during aging effectively rescues all of these phenotypes. These results indicate that the Drosophila model recapitulates the age-dependent cognitive decline observed in humans. This places Fragile X in a category with several other diseases that result in age-dependent cognitive decline. This demonstrates a role for the Drosophila Fragile X Mental Retardation Protein (dFMR1) in neuronal physiology with regard to cognition during the aging process. Our results indicate that misregulation of mGluR activity may be causative of this age onset decline and strengthens the possibility that mGluR antagonists and lithium may be potential pharmacologic compounds for counteracting several Fragile X symptoms.
PMCID: PMC2866528  PMID: 20039205
Drosophila; Age onset cognitive deficits; Fragile X; Metabotropic glutamate receptor; Courtship conditioning assays; Pharmacological rescue
10.  Pharmacological and genetic reversal of age dependent cognitive deficits due to decreased presenilin function 
Alzheimer's disease is the leading cause of cognitive loss and neurodegeneration in the developed world. Although its genetic and environmental causes are not generally known, familial forms of the disease (FAD) are due to mutations in a single copy of the Presenilin (PS) and Amyloid Precursor Protein (APP) genes. The dominant inheritance pattern of FAD indicates that it may be due to gain or change of function mutations. Studies of FAD-linked forms of presenilin in model organisms, however, indicate that they are loss of function, leading to the possibility that a reduction in PS activity might contribute to FAD and that proper psn levels are important for maintaining normal cognition throughout life. To explore this issue further, we have tested the effect of reducing psn activity during aging in Drosophila melanogaster males. We have found that flies in which the dosage of psn function is reduced by 50% display age-onset impairments in learning and memory. Treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium during the aging process prevented the onset of these deficits, and treatment of aged flies reversed the age-dependent deficits. Genetic reduction of DmGluRA, the inositol trisphosphate receptor (InsP3R) or IPPase also prevented these age-onset cognitive deficits. These findings suggest that reduced psn activity may contribute to the age onset cognitive loss observed with FAD. They also indicate that enhanced mGluR signaling and calcium release regulated by InsP3R as underlying causes of the age-dependent cognitive phenotypes observed when psn activity is reduced.
PMCID: PMC2917645  PMID: 20631179
Presenilin; Age onset cognitive deficits; Alzheimer’s disease; Metabotropic glutamate receptor; Inositol trisphosphate receptor; Drosophila
11.  Short and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein 
The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. We show that two isoforms of D. melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation.
PMCID: PMC2880182  PMID: 20463240
FMRP; memory; alternative splicing; protein isoform; prion-related domain; RNA-binding
12.  Paucity of Clinical Disease despite Serological Autoimmunity and Kidney Pathology in Lupus-Prone New Zealand Mixed 2328 Mice Deficient in BAFF1 
Constitutive overexpression of B cell-activating factor belonging to the TNF family (BAFF) promotes development of systemic lupus erythematosus (SLE), and treatment of SLE mice with BAFF antagonists ameliorates disease. To determine whether SLE can develop de novo in BAFF-deficient hosts, BAFF-deficient New Zealand Mixed (NZM) 2328 (NZM.Baff−/−) mice were generated. In NZM.Baff−/− mice, spleen B cells (including CD5+ B1a and CD5− B1b B cells), germinal centers, Ig-secreting cells, and T cells were reduced in comparison to NZM.Baff+/+ mice. Serum total Ig and autoantibody levels were reduced at 4–6 mo but approached wild-type levels with increasing age, indicating that autoreactive B cells can survive and secrete autoantibodies despite the complete absence of BAFF. At least some of these autoantibodies are nephrophilic in that glomerular deposition of total IgG and IgG1 (but not of IgG2a, IgG2b, or C3) was substantial in NZM.Baff−/− mice by 12–13 mo of age. Despite proliferative glomerulonephritis, highlighted by widespread glomerular hyaline thrombi, being common among NZM.Baff−/− mice by 6–7 mo of age, severe proteinuria and mortality were greatly attenuated. These results demonstrate that the lifelong absence of BAFF does not protect NZM 2328 mice from serological autoimmunity and renal pathology. Nevertheless, the character of the renal pathology is altered, and the mice are largely spared from clinically overt disease (severe proteinuria and premature death). These observations may have profound ramifications for the use of BAFF antagonists in human SLE and related diseases.
PMCID: PMC2896675  PMID: 16888029

Results 1-12 (12)