Resting State fMRI Reveals Diminished Functional Connectivity in a Mouse Model of Amyloidosis

Introduction

Functional connectivity (FC) studies have gained immense popularity in the evaluation of several neurological disorders, such as Alzheimer’s disease (AD). AD is a complex disorder, characterised by several pathological features. The problem with FC studies in patients is that it is not straightforward to focus on a specific aspect of pathology. In the current study, resting state functional magnetic resonance imaging (rsfMRI) is applied in a mouse model of amyloidosis to assess the effects of amyloid pathology on FC in the mouse brain.

Methods

Nine APP/PS1 transgenic and nine wild-type mice (average age 18.9 months) were imaged on a 7T MRI system. The mice were anesthetized with medetomidine and rsfMRI data were acquired using a gradient echo EPI sequence. The data were analysed using a whole brain seed correlation analysis and interhemispheric FC was evaluated using a pairwise seed analysis. Qualitative histological analyses were performed to assess amyloid pathology, inflammation and synaptic deficits.

Results

The whole brain seed analysis revealed an overall decrease in FC in the brains of transgenic mice compared to wild-type mice. The results showed that interhemispheric FC was relatively preserved in the motor cortex of the transgenic mice, but decreased in the somatosensory cortex and the hippocampus when compared to the wild-type mice. The pairwise seed analysis confirmed these results. Histological analyses confirmed the presence of amyloid pathology, inflammation and synaptic deficits in the transgenic mice.

Conclusions

In the current study, rsfMRI demonstrated decreased FC in APP/PS1 transgenic mice compared to wild-type mice in several brain regions. The APP/PS1 transgenic mice had advanced amyloid pathology across the brain, as well as inflammation and synaptic deficits surrounding the amyloid plaques. Future studies should longitudinally evaluate APP/PS1 transgenic mice and correlate the rsfMRI findings to specific stages of amyloid pathology.     

Modeling Alzheimer’s Disease in Mouse without Mutant Protein Overexpression: Cooperative and Independent Effects of Aβ and Tau

Background

Alzheimer’s disease (AD), the most common cause of dementia in the elderly, has two pathological hallmarks: Aβ plaques and aggregation of hyperphosphorylated tau (p-tau). Aβ is a cleavage product of Amyloid Precursor Protein (APP). Presenilin 1 (PS1) and presenilin 2 (PS2) are the catalytic subunit of γ-secretase, which cleaves APP and mediates Aβ production. Genetic mutations in APP, PSEN1 or PSEN2 can lead to early onset of familial AD (FAD). Although mutations in the tau encoding gene MAPT leads to a subtype of frontotemporal dementia and these mutations have been used to model AD tauopathy, no MAPT mutations have been found to be associated with AD.

Results

To model AD pathophysiology in mice without the gross overexpression of mutant transgenes, we created a humanized AD mouse model by crossing the APP and PSEN1 FAD knock-in mice with the htau mice which express wildtype human MAPT genomic DNA on mouse MAPT null background (APP/PS1/htau). The APP/PS1/htau mice displayed mild, age-dependent, Aβ plaques and tau hyperphosphorylation, thus successfully recapitulating the late-onset AD pathological hallmarks. Selected biochemical analyses, including p-tau western blot, γ-secretase activity assay, and Aβ ELISA, were performed to study the interaction between Aβ and p-tau. Subsequent behavioral studies revealed that the APP/PS1/htau mice showed reduced mobility in old ages and exaggerated fear response. Genetic analysis suggested that the fear phenotype is due to a synergic interaction between Aβ and p-tau, and it can be completely abolished by tau deletion.

Conclusion

The APP/PS1/htau model represents a valuable and disease-relevant late-onset pre-clinical AD animal model because it incorporates human AD genetics without mutant protein overexpression. Analysis of the mice revealed both cooperative and independent effects of Aβ and p-tau.     

Increased neointimal thickening in dystrophin-deficient mdx mice

Background

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation.

Methodology/Principal Findings

We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice.

Conclusions/Significance

These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.     

L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle

Background

In dystrophic mdx skeletal muscle, aberrant Ca²⁺ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca²⁺ currents. In regenerating mdx muscle, ‘revertant’ fibres restore dystrophin expression. Their functionality involving DHPR-Ca²⁺-channels is elusive.

Methods and Results

We developed a novel ‘in-situ’ confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified ‘mdx-like’. Some mdx fibres, however, had strong ‘wt-like’ dystrophin signals and were identified as ‘revertants’. Split mdx fibres were mostly ‘mdx-like’ and are not generally ‘revertants’. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in ‘revertants’. Using the two-micro-electrode-voltage clamp technique, Ca²⁺-current amplitudes (i_max) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely ‘revertants’, with amplitudes similar to wt or MinD fibres. Ca²⁺ current activation curves were similar in ‘wt-like’ and ‘mdx-like’ aged mdx fibres and are not the cause for the differences in current amplitudes. i_max amplitudes were fully restored in MinD fibres.

Conclusions

We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and ‘revertant’ mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated ‘revertant’ fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD.     

Development and Notch signaling requirements of the zebrafish choroid plexus

Background

The choroid plexus (CP) is an epithelial and vascular structure in the ventricular system of the brain that is a critical part of the blood-brain barrier. The CP has two primary functions, 1) to produce and regulate components of the cerebral spinal fluid, and 2) to inhibit entry into the brain of exogenous substances. Despite its importance in neurobiology, little is known about how this structure forms.

Methodology and Principal Findings

Here we show that the transposon-mediated enhancer trap zebrafish line Et^Mn16 expresses green fluorescent protein within a population of cells that migrate toward the midline and coalesce to form the definitive CP. We further demonstrate the development of the integral vascular network of the definitive CP. Utilizing pharmacologic pan-notch inhibition and specific morpholino-mediated knockdown, we demonstrate a requirement for Notch signaling in choroid plexus development. We identify three Notch signaling pathway members as mediating this effect, notch1b, deltaA, and deltaD.

Conclusions and Significance

This work is the first to identify the zebrafish choroid plexus and to characterize its epithelial and vasculature integration. This study, in the context of other comparative anatomical studies, strongly indicates a conserved mechanism for development of the CP. Finally, we characterize a requirement for Notch signaling in the developing CP. This establishes the zebrafish CP as an important new system for the determination of key signaling pathways in the formation of this essential component of the vertebrate brain.     

Inhibiting AKT Phosphorylation Employing Non-Cytotoxic Anthraquinones Ameliorates TH2 Mediated Allergic Airways Disease and Rhinovirus Exacerbation

Background

Severe asthma is associated with T helper (T_H) 2 and 17 cell activation, airway neutrophilia and phosphoinositide-3-kinase (PI3K) activation. Asthma exacerbations are commonly caused by rhinovirus (RV) and also associated with PI3K-driven inflammation. Anthraquinone derivatives have been shown to reduce PI3K-mediated AKT phosphorylation in-vitro.

Objective

To determine the anti-inflammatory potential of anthraquinones in-vivo.

Methods

BALB/c mice were sensitized and challenged with crude house dust mite extract to induce allergic airways disease and treated with mitoxantrone and a novel non-cytotoxic anthraquinone derivative. Allergic mice were also infected with RV1B to induce an exacerbation.

Results

Anthraquinone treatment reduced AKT phosphorylation, hypoxia-inducible factor-1α and vascular endothelial growth factor expression, and ameliorated allergen- and RV-induced airways hyprereactivity, neutrophilic and eosinophilic inflammation, cytokine/chemokine expression, mucus hypersecretion, and expression of T_H2 proteins in the airways. Anthraquinones also boosted type 1 interferon responses and limited RV replication in the lung.

Conclusion

Non-cytotoxic anthraquinone derivatives may be of therapeutic benefit for the treatment of severe and RV-induced asthma by blocking pro-inflammatory pathways regulated by PI3K/AKT.     

Blueberry and Mulberry Juice Prevent Obesity Development in C57BL/6 Mice

Objectives

To establish whether blueberry (Vaccinium ashei) and mulberry (Morus australis Poir) juice, anthocyanin rich fruit juice, may help counteract obesity.

Design

And Methods: Four-week-old C57BL/6 mice were fed a high-fat diet (HFD) with or without blueberry and mulberry juice for 12 weeks. Body weight, serum and hepatic lipids, liver and adipose tissues morphology, insulin and leptin were assessed.

Results

Mice fed HFD exhibited increased body weight, insulin resistance, serum and hepatic lipids. In comparison, blueberry and mulberry juice inhibited body weight gain, decreased the serum cholesterol, reduced the resistance to insulin, attenuated lipid accumulation and decreased the leptin secretin.

Conclusion

These results indicate that blueberry and mulberry juice may help counteract obesity.     

No Evidence of Persisting Unrepaired Nuclear DNA Single Strand Breaks in Distinct Types of Cells in the Brain,Kidney, and Liver of Adult Mice after Continuous Eight-Week 50 Hz Magnetic Field Exposure with Flux Density of 0.1 mT or 1.0 mT

Background

It has been hypothesized in the literature that exposure to extremely low frequency electromagnetic fields (50 or 60 Hz) may lead to human health effects such as childhood leukemia or brain tumors. In a previous study investigating multiple types of cells from brain and kidney of the mouse (Acta Neuropathologica 2004; 107: 257–264), we found increased unrepaired nuclear DNA single strand breaks (nDNA SSB) only in epithelial cells of the choroid plexus in the brain using autoradiographic methods after a continuous eight-week 50 Hz magnetic field (MF) exposure of adult mice with flux density of 1.5 mT.

Methods

In the present study we tested the hypothesis that MF exposure with lower flux densities (0.1 mT, i.e., the actual exposure limit for the population in most European countries, and 1.0 mT) shows similar results to those in the previous study. Experiments and data analysis were carried out in a similar way as in our previous study.

Results

Continuous eight-week 50 Hz MF exposure with 0.1 mT or 1.0 mT did not result in increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice. MF exposure with 1.0 mT led to reduced unscheduled DNA synthesis (UDS) in epithelial cells in the choroid plexus of the fourth ventricle in the brain (EC-CP) and epithelial cells of the cortical collecting duct in the kidney, as well as to reduced mtDNA synthesis in neurons of the caudate nucleus in the brain and in EC-CP.

Conclusion

No evidence was found for increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice after continuous eight-week 50 Hz magnetic field exposure with flux density of 0.1 mT or 1.0 mT.     

Infection with Porphyromonas gingivalis Exacerbates Endothelial Injury in Obese Mice

Background

A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity.

Methods

In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression.

Results

Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate.

Conclusions

Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.     

Cellular,Molecular and Functional Characterisation of YAC Transgenic Mouse Models of Friedreich Ataxia

Background

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, caused by a GAA repeat expansion mutation within intron 1 of the FXN gene. We have previously established and performed preliminary characterisation of several human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing GAA repeat expansions, Y47R (9 GAA repeats), YG8R (90 and 190 GAA repeats) and YG22R (190 GAA repeats).

Methodology/Principal Findings

We now report extended cellular, molecular and functional characterisation of these FXN YAC transgenic mouse models. FXN transgene copy number analysis of the FRDA mice demonstrated that the YG22R and Y47R lines each have a single copy of the FXN transgene while the YG8R line has two copies. Single integration sites of all transgenes were confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We identified significant functional deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8R and YG22R FRDA mice compared to Y47R and wild-type control mice. We also confirmed increased somatic GAA repeat instability in the cerebellum and brain of YG22R and YG8R mice, together with significantly reduced levels of FXN mRNA and protein in the brain and liver of YG8R and YG22R compared to Y47R.

Conclusions/Significance

Together these studies provide a detailed characterisation of our GAA repeat expansion-based YAC transgenic FRDA mouse models that will help investigations of FRDA disease mechanisms and therapy.     

Intestine-Specific Deletion of Microsomal Triglyceride Transfer Protein Increases Mortality in Aged Mice

Background

Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8–10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis.

Methods

Aged (20–24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival.

Results

In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-X_L ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice.

Conclusions

Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice.     

Response-Predictive Gene Expression Profiling of Glioma Progenitor Cells In Vitro

Background

High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist.

Methods

In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a) to enrich specimens for brain tumor initiating cells and (b) to confront cells with a therapeutic agent before expression profiling.

Results

As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.

Conclusion

For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.     

In vivo MRI characterization of progressive cardiac dysfunction in the mdx mouse model of muscular dystrophy

Aims

The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice.

Methods and Results

Serial measurements of cardiac morphology and function were made in the same group of mdx mice and controls (housed in a non-SPF facility) using MRI at 1, 3, 6, 9 and 12 months after birth. Left ventricular (LV) and right ventricular (RV) systolic and diastolic function, response to dobutamine stress and myocardial fibrosis were assessed. RV dysfunction preceded LV dysfunction, with RV end systolic volumes increased and RV ejection fractions reduced at 3 months of age. LV ejection fractions were reduced at 12 months, compared with controls. An abnormal response to dobutamine stress was identified in the RV of mdx mice as early as 1 month. Late-gadolinium-enhanced MRI identified increased levels of myocardial fibrosis in 6, 9 and 12-month-old mdx mice, the extent of fibrosis correlating with the degree of cardiac remodeling and hypertrophy.

Conclusions

MRI could identify cardiac abnormalities in the RV of mdx mice as young as 1 month, and detected myocardial fibrosis at 6 months. We believe these to be the earliest MRI measurements of cardiac function reported for any mice, and the first use of late-gadolinium-enhancement in a mouse model of congenital cardiomyopathy. These techniques offer a sensitive and clinically relevant in vivo method for assessment of cardiomyopathy caused by muscular dystrophy and other diseases.     

Bone Marrow-Derived Microglia Infiltrate into the Paraventricular Nucleus of Chronic Psychological Stress-Loaded Mice

Background

Microglia of the central nervous system act as sentinels and rapidly react to infection or inflammation. The pathophysiological role of bone marrow-derived microglia is of particular interest because they affect neurodegenerative disorders and neuropathic pain. The hypothesis of the current study is that chronic psychological stress (chronic PS) induces the infiltration of bone marrow-derived microglia into hypothalamus by means of chemokine axes in brain and bone marrow.

Methods and Findings

Here we show that bone marrow-derived microglia specifically infiltrate the paraventricular nucleus (PVN) of mice that received chronic PS. Bone marrow derived-microglia are CX₃CR1^lowCCR2⁺CXCR4^high, as distinct from CX₃CR1^highCCR2^-CXCR4^low resident microglia, and express higher levels of interleukin-1β (IL-1β) but lower levels of tumor necrosis factor-α (TNF-α). Chronic PS stimulates the expression of monocyte chemotactic protein-1 (MCP-1) in PVN neurons, reduces stromal cell-derived factor-1 (SDF-1) in the bone marrow and increases the frequency of CXCR4⁺ monocytes in peripheral circulation. And then a chemokine (C-C motif) receptor 2 (CCR2) or a β₃-adrenoceptor blockade prevents infiltration of bone marrow-derived microglia in the PVN.

Conclusion

Chronic PS induces the infiltration of bone marrow-derived microglia into PVN, and it is conceivable that the MCP-1/CCR2 axis in PVN and the SDF-1/CXCR4 axis in bone marrow are involved in this mechanism.     

Isolation of Irkut Virus from a Murina leucogaster Bat in China

Background and objectives

Bats are recognized as a major reservoir of lyssaviruses; however, no bat lyssavirus has been isolated in Asia except for Aravan and Khujand virus in Central Asia. All Chinese lyssavirus isolates in previous reports have been of species rabies virus, mainly from dogs. Following at least two recent bat-associated human rabies-like cases in northeast China, we have initiated a study of the prevalence of lyssaviruses in bats in Jilin province and their public health implications. A bat lyssavirus has been isolated and its pathogenicity in mice and genomic alignment have been determined.

Results

We report the first isolation of a bat lyssavirus in China, from the brain of a northeastern bat, Murina leucogaster. Its nucleoprotein gene shared 92.4%/98.9% (nucleotide) and 92.2%/98.8% (amino acid) identity with the two known Irkut virus isolates from Russia, and was designated IRKV-THChina12. Following intracranial and intramuscular injection, IRKV-THChina12 produced rabies-like symptoms in adult mice with a short inoculation period and high mortality. Nucleotide sequence analysis showed that IRKV-THChina12 has the same genomic organization as other lyssaviruses and its isolation provides an independent origin for the species IRKV.

Conclusions

We have identified the existence of a bat lyssavirus in a common Chinese bat species. Its high pathogenicity in adult mice suggests that public warnings and medical education regarding bat bites in China should be increased, and that surveillance be extended to provide a better understanding of Irkut virus ecology and its significance for public health.     

Genetic Variation in SULF2 Is Associated with Postprandial Clearance of Triglyceride-Rich Remnant Particles and Triglyceride Levels in Healthy Subjects

Context

Nonfasting (postprandial) triglyceride concentrations have emerged as a clinically significant cardiovascular disease risk factor that results from accumulation of remnant triglyceride-rich lipoproteins (TRLs) in the circulation. The remnant TRLs are cleared from the circulation by hepatic uptake, but the specific mechanisms involved are unclear. The syndecan-1 heparan sulfate proteoglycan (HSPG) pathway is important for the hepatic clearance of remnant TRLs in mice, but its relevance in humans is unclear.

Objective

We sought to determine whether polymorphisms of the genes responsible for HSPG assembly and disassembly contribute to atherogenic dyslipoproteinemias in humans.

Patients And Design

We performed an oral fat load in 68 healthy subjects. Lipoproteins (chylomicrons and very low density lipoproteins 1 and 2) were isolated from blood, and the area under curve and incremental area under curve for postprandial variables were calculated. Single nucleotide polymorphisms in genes encoding syndecan-1 and enzymes involved in the synthesis or degradation of HSPG were genotyped in the study subjects.

Results

Our results indicate that the genetic variation rs2281279 in SULF2 associates with postprandial clearance of remnant TRLs and triglyceride levels in healthy subjects. Furthermore, the SNP rs2281279 in SULF2 associates with hepatic SULF2 mRNA levels.

Conclusions

In humans, mild but clinically relevant postprandial hyperlipidemia due to reduced hepatic clearance of remnant TRLs may result from genetic polymorphisms that affect hepatic HSPG.     

Inhibition of Elongation Factor-2 Kinase Augments the Antitumor Activity of Temozolomide against Glioma

Background

Glioblastoma multiforme (GBM), the most common form of brain cancer with an average survival of less than 12 months, is a highly aggressive and fatal disease characterized by survival of glioma cells following initial treatment, invasion through the brain parenchyma and destruction of normal brain tissues, and ultimately resistance to current treatments. Temozolomide (TMZ) is commonly used chemotherapy for treatment of primary and recurrent high-grade gliomas. Nevertheless, the therapeutic outcome of TMZ is often unsatisfactory. In this study, we sought to determine whether eEF-2 kinase affected the sensitivity of glioma cells to treatment with TMZ.

Methodology/Principal Findings

Using RNA interference approach, a small molecule inhibitor of eEF-2 kinase, and in vitro and in vivo glioma models, we observed that inhibition of eEF-2 kinase could enhance sensitivity of glioma cells to TMZ, and that this sensitizing effect was associated with blockade of autophagy and augmentation of apoptosis caused by TMZ.

Conclusions/Significance

These findings demonstrated that targeting eEF-2 kinase can enhance the anti-glioma activity of TMZ, and inhibitors of this kinase may be exploited as chemo-sensitizers for TMZ in treatment of malignant glioma.     

Role of Mental Retardation-Associated Dystrophin-Gene Product Dp71 in Excitatory Synapse Organization,Synaptic Plasticity and Behavioral Functions

Background

Duchenne muscular dystrophy (DMD) is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR), likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood.

Methodology/Principal Findings

Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory.

Conclusions/Significance

Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.     

Sirtuin 1 (SIRT1) Activation Mediates Sildenafil Induced Delayed Cardioprotection against Ischemia-Reperfusion Injury in Mice

Background

It has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury.

Objective/Hypothesis

We tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1.

Methods

Adult male ICR mice were treated with SIL (0.7 mg/kg, i.p.), Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control), or saline (0.2 mL). The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis.

Results

Both SIL- and RSV-treated mice had increased cardiac SIRT1 activity (P<0.001) as compared to the saline-treated controls 24 hours after drug treatment. In isolated ventricular cardiomyocytes, pretreatment with SIL (1 µM) or RSV (1 µM) for one hour in vitro also upregulated SIRT1 activity (P<0.05). We further examined the causative relationship between SIRT1 activation and SIL-induced late cardioprotection. Pretreatment with SIL (or RSV) 24 hours prior to 30 min ischemia and 24 hours of reperfusion significantly reduced infarct size, which was associated with a significant increase in SIRT1 activity (P<0.05). Moreover, sirtinol (a SIRT1 inhibitor, 5 mg/kg, i.p.) given 30 min before I-R blunted the infarct-limiting effect of SIL and RSV (P<0.001).

Conclusion

Our study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications.