Blockade of EphA4 signaling ameliorates hippocampal synaptic dysfunctions in mouse models of Alzheimer’s disease

Alzheimer’s disease (AD), characterized by cognitive decline, has emerged as a disease of synaptic failure. The present study reveals an unanticipated role of erythropoietin-producing hepatocellular A4 (EphA4) in mediating hippocampal synaptic dysfunctions in AD and demonstrates that blockade of the ligand-binding domain of EphA4 reverses synaptic impairment in AD mouse models. Enhanced EphA4 signaling was observed in the hippocampus of amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model of AD, whereas soluble amyloid-β oligomers (Aβ), which contribute to synaptic loss in AD, induced EphA4 activation in rat hippocampal slices. EphA4 depletion in the CA1 region or interference with EphA4 function reversed the suppression of hippocampal long-term potentiation in APP/PS1 transgenic mice, suggesting that the postsynaptic EphA4 is responsible for mediating synaptic plasticity impairment in AD. Importantly, we identified a small-molecule rhynchophylline as a novel EphA4 inhibitor based on molecular docking studies. Rhynchophylline effectively blocked the EphA4-dependent signaling in hippocampal neurons, and oral administration of rhynchophylline reduced the EphA4 activity effectively in the hippocampus of APP/PS1 transgenic mice. More importantly, rhynchophylline administration restored the impaired long-term potentiation in transgenic mouse models of AD. These findings reveal a previously unidentified role of EphA4 in mediating AD-associated synaptic dysfunctions, suggesting that it is a new therapeutic target for this disease.Cognitive impairment, regarded as an early manifestation of Alzheimer’s disease (AD), is attributable to disruptions of synaptic functions which correlate with the severity of memory deficit in AD (1). Soluble amyloid-β peptide oligomers (Aβ), which are generated by the proteolytic cleavage of amyloid precursor protein (APP), are believed to be a major causative agent of synaptic impairment during AD progression (2). Thus, reversing Aβ-induced synaptic deficits is considered a promising therapeutic approach for alleviating cognitive impairment in AD (3).Aβ binds to synaptic sites (4), resulting in synaptic loss and reduced glutamatergic synaptic transmission (5, 6). Aβ also rapidly impairs synaptic plasticity in the hippocampus; this includes the inhibition of long-term potentiation (LTP) (2) and facilitation of long-term depression (LTD) (7), which are major cellular mechanisms associated with learning and memory. Synaptic defects triggered by Aβ are mediated by the internalization and down-regulation of both NMDA- and AMPA-type glutamate receptors (8, 9) together with a reduction of dendritic spines (6), where excitatory synapses are located. Therefore, identifying molecular targets that mediate the action of Aβ in synaptic depression in AD is crucial for the development of therapeutic interventions for AD. Interestingly, various cell surface receptors such as α7-nicotinic acetylcholine receptors, metabotropic glutamate receptors, insulin receptors, and the receptor tyrosine kinase, EphB2, are reported to mediate the action of Aβ at synapses (10).The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases is important for the regulation of synapse development and synaptic plasticity (11, 12). EphB enhances synapse development via its interaction with NMDA receptors (13), whereas EphA4, which is mainly expressed in the adult hippocampus, acts as a negative regulator of neurotransmission and hippocampal synaptic plasticity (14). EphA4 activation by its ligands, ephrins, triggers forward signaling (12) that leads to the retraction of dendritic spines via cyclin-dependent kinase 5 (Cdk5)-dependent RhoA activation and reduced cell adhesion (15–17). EphA4 also causes the removal of synaptic and surface AMPA receptors during homeostatic plasticity (18, 19). Interestingly, AD patients with only mild cognitive deficits exhibit deregulated EphB and EphA4 expression (20). Given that EphA4 activation results in dendritic spine loss and reduced AMPA receptor abundance (14, 19, 21), which are potential mechanisms that underlie synaptic dysfunctions in AD (6, 8), we investigated the possible link between EphA4 signaling and Aβ-induced synaptic failure.The present study demonstrates that EphA4 mediates the Aβ-induced impairment of synaptic plasticity. Depletion of postsynaptic EphA4 or blockade of the activity of EphA4 through targeting its ligand-binding domain reversed the synaptic deficits in AD mouse models. Importantly, molecular docking analysis identified a small molecule, rhynchophylline (Rhy), as a candidate EphA4 inhibitor. Rhy rescued the impaired neurotransmission induced by Aβ as well as the LTP defects in the AD mouse models. Thus, the present findings not only reveal an important role of EphA4 in the pathogenesis of AD, but also identify a small-molecule inhibitor of EphA4 that can be further developed as a potential therapeutic intervention for AD.     

Brain cellular senescence in mouse models of Alzheimer’s disease

The accumulation of senescent cells contributes to aging pathologies, including neurodegenerative diseases, and its selective removal improves physiological and cognitive function in wild-type mice as well as in Alzheimer’s disease (AD) models. AD models recapitulate some, but not all components of disease and do so at different rates. Whether brain cellular senescence is recapitulated in some or all AD models and whether the emergence of cellular senescence in AD mouse models occurs before or after the expected onset of AD-like cognitive deficits in these models are not yet known. The goal of this study was to identify mouse models of AD and AD-related dementias that develop measurable markers of cellular senescence in brain and thus may be useful to study the role of cellular senescence in these conditions. We measured the levels of cellular senescence markers in the brains of P301S(PS19), P301L, hTau, and 3xTg-AD mice that model amyloidopathy and/or tauopathy in AD and related dementias and in wild-type, age-matched control mice for each strain. Expression of cellular senescence markers in brains of transgenic P301L and 3xTg-AD mice was largely indistinguishable from that in WT control age-matched mice. In contrast, markers of cellular senescence were differentially increased in brains of transgenic hTau and P301S(PS19) mice as compared to WT control mice before the onset of AD-like cognitive deficits. Taken together, our data suggest that P301S(PS19) and hTau mice may be useful models for the study of brain cellular senescence in tauopathies including, but not limited to, AD.

     

Reversal of hyperglycemia in diabetic mouse models using induced-pluripotent stem (iPS)-derived pancreatic β-like cells

Diabetes mellitus is characterized by either the inability to produce insulin (type 1 diabetes) or as insensitivity to insulin secreted by the body (type 2 diabetes). In either case, the body is unable to move blood glucose efficiently across cell membranes to be used. This leads to a variety of local and systemic detrimental effects. Current treatments for diabetes focus on exogenous insulin administration and dietary control. Here, we describe a potential cure for diabetes using a cellular therapy to ameliorate symptoms associated with both reduced insulin secretion and insulin sensitivity. Using induced pluripotent stem (iPS) cells, we were able to derive β-like cells similar to the endogenous insulin-secreting cells in mice. These β-like cells secreted insulin in response to glucose and corrected a hyperglycemic phenotype in two mouse models of type 1 and 2 diabetes via an iPS cell transplant. Long-term correction of hyperglycemia was achieved, as determined by blood glucose and hemoglobin A1c levels. These data provide an initial proof of principle for potential clinical applications of reprogrammed somatic cells in the treatment of diabetes type 1 or 2.     

Over-nutrition, obesity and insulin resistance in the development of β-cell dysfunction

The incidence of type 2 diabetes mellitus (DM2) has increased dramatically over the last several decades, largely driven by equally worrisome growing rates of obesity. Chronic diabetic complications are leading causes of morbidity and mortality worldwide. Key players in the pathophysiology of DM2 are insulin resistance and β cell dysfunction, which in turn is a result of both β cell functional abnormality as well as reduced β cell mass. The mechanisms implicated are multifactorial and include genetic and environmental factors related to obesity. Glucose homeostasis is critically dependent on a finely regulated balance between insulin sensitivity and output in the pancreas, and insulin resistance demands a corresponding rise in insulin output in order to maintain normal glycemia. However, this compensation is lost in individuals predisposed to DM2, resulting in overt hyperglycemia. Furthermore, insulin resistance related to excess adiposity is linked to several abnormalities which impact β cell function and viability. These include glucotoxicity, lipotoxicity, increased oxidative stress, and inflammation. In addition, insulin signaling in the β cell is essential to its own functionality and viability, and obesity-related abnormalities in insulin signaling are known to induce failure of insulin secretion and hyperglycemia. Insulin resistance in the β cell arises from defects in phosphorylation/activation of insulin receptor substrates (IRS) proteins, which result in impairment in glucose sensing, glucose stimulated insulin secretion, and also in increased loss of β cells. This review intends to provide an update on the main characteristics and mechanisms that link obesity and insulin resistance to β cell dysfunction in the pathogenesis of DM2.     

Role of skeletal muscle lipids in the pathogenesis of insulin resistance of obesity and type 2 diabetes

Obesity predisposes individuals to the development of insulin resistance, which is a risk factor for type 2 diabetes, and muscle plays a central role in this phenomenon. Insulin resistance is associated with: (i) a metabolic inflexibility characterized by a reduced impaired switching from free fatty acid (FA) to carbohydrate substrates; and (ii) an ectopic accumulation of triglyceride in skeletal muscle, generating a cellular “lipotoxicity”, but triglyceride per se, does not contribute to insulin resistance (“athlete’s paradox”). A large body of evidence supports the idea that a decreased mitochondrial capacity to oxidize FA leads to an accretion of intracellular triglyceride and an accumulation of acyl-CoAs, which are used to synthesize diacylglycerol and ceramide. These lipid derivatives activate serine kinases, leading to increase of insulin receptor substrate 1 serine phosphorylation, which impairs insulin signaling. A second model proposes that insulin resistance arises from an excessive mitochondrial FA oxidation. Studies have shown that the type of FA, unsaturated or saturated, is critical in the development of insulin resistance. It should be also stressed that FA oversupply activates inflammatory signals, induces endoplasmic reticulum stress, increases mitochondrial oxidative stress and influences the regulation of genes that contributes to impaired glucose metabolism. These cellular insults are thought to engage stress-sensitive serine kinases disrupting insulin signaling. In conclusion, reduced dietary lipid intake in association with physical exercise could be a therapeutic option to improve insulin sensitivity.     

The diversity of sex steroid action: novel functions of hydroxysteroid (17β) dehydrogenases as revealed by genetically modified mouse models

Disturbed action of sex steroid hormones, i.e. androgens and estrogens, is involved in the pathogenesis of various severe diseases in humans. Interestingly, recent studies have provided data further supporting the hypothesis that the circulating hormone concentrations do not explain all physiological and pathological processes observed in hormone-dependent tissues, while the intratissue sex steroid concentrations are determined by the expression of steroid metabolising enzymes in the neighbouring cells (paracrine action) and/or by target cells themselves (intracrine action). This local sex steroid production is also a valuable treatment option for developing novel therapies against hormonal diseases. Hydroxysteroid (17β) dehydrogenases (HSD17Bs) compose a family of 14 enzymes that catalyse the conversion between the low-active 17-keto steroids and the highly active 17β-hydroxy steroids. The enzymes frequently expressed in sex steroid target tissues are, thus, potential drug targets in order to lower the local sex steroid concentrations. The present review summarises the recent data obtained for the role of HSD17B1, HSD17B2, HSD17B7 and HSD17B12 enzymes in various metabolic pathways and their physiological and pathophysiological roles as revealed by the recently generated genetically modified mouse models. Our data, together with that provided by others, show that, in addition to having a role in sex steroid metabolism, several of these HSD17B enzymes possess key roles in other metabolic processes: for example, HD17B7 is essential for cholesterol biosynthesis and HSD17B12 is involved in elongation of fatty acids. Additional studies in vitro and in vivo are to be carried out in order to fully define the metabolic role of the HSD17B enzymes and to evaluate their value as drug targets.     

The effect of the deterioration of insulin sensitivity onβ-cell function in growth-hormone-deficient adults following 4-month growth hormone replacement therapy

The purpose of the present study was to evaluate the combined effect of GH treatment on body composition and glucose metabolism, with special focus on β-cell function in adult GHD patients. In a double-blind placebo-controlled design, 24 GHD adults (18M/6F), were randomized to 4 months treatment with biosynthetic GH 2 IU/m²s.c. daily (n=13) or placebo (n=11). At inclusion and 4 months later an oral glucose tolerance test (OGTT), a frequently sampled intravenous glucose tolerance test (FSIGT) and dual-energy X-ray absorptiometry (DXA) whole-body scanning were performed. During the study period, body weight decreased 1.6 kg from 94.0 ± 18.7 to 92.4 ± 19.4 kg (mean ± SD) (P<0.05) in the GH-treated group, but remained unchanged in the placebo group. Fat mass decreased from 32.4 ± 9.6 to 28.1 ± 10.5 kg (P<0.001), whereas lean body mass increased from 58.3 ± 11.5 to 61.0 ± 11.7 kg (P<0.01) in the GH-treated group. Treatment with GH for 4 months resulted in a significant increase in fasting blood glucose (before GH 5.0 ± 0.3 and after 5.4 ± 0.6 mmol/l,P<0.05), fasting plasma insulin (before GH 38.4 ± 30.2 and after 55.3 ± 34.7 pmol/l,P<0.02) and fasting proinsulin (before 8.1 ± 6.7 and after 14.6 ± 16.1 pmol/l,P<0.05). The insulin sensitivity index S_I, estimated by Bergmans Minimal Model, decreased significantly [before GH 1.1 ± 0.7 and after 0.4 ± 0.2 10^–4(min × pmol/l),P<0.003]. The non-insulin-dependent glucose uptake (glucose effectiveness S_Gdid not change (before GH 0.017 ± 0.005 and after 0.015 ± 0.006 min^–1, NS). Insulin secretion was enhanced during GH therapy, but insufficiently to match the changes in S_I, resulting in a higher blood glucose level during an OGTT. Blood glucose at 120 min was 5.5 and 6.3 mmol/l before and after GH treatment, respectively (P= 0.07). One patient developed impaired glucose tolerance.Short-term GH replacement therapy in a dose of about 2 IU/m²daily in GHD adults induces a reduction in insulin sensitivity, despite favourable changes in body composition, and an inadequate enhancement of insulin secretion.     

Anti-inflammatory effects of yerba maté extract (Ilex paraguariensis) ameliorate insulin resistance in mice with high fat diet-induced obesity

The aim of the present study was to evaluate the effects of yerba maté extract upon markers of insulin resistance and inflammatory markers in mice with high fat diet-induced obesity. The mice were introduced to either standard or high fat diets. After 12 weeks on a high fat diet, mice were randomly assigned to one of the two treatment conditions, water or yerba maté extract at 1.0 gkg(-1). After treatment, glucose blood level and hepatic and soleus muscle insulin response were evaluated. Serum levels of TNF-α and IL-6 were evaluated by ELISA, liver tissue was examined to determine the mRNA levels of TNF-α, IL-6 and iNOS, and the nuclear translocation of NF-κB was determined by an electrophoretic mobility shift assay. Our data show improvements in both the basal glucose blood levels and in the response to insulin administration in the treated animals. The molecular analysis of insulin signalling revealed a restoration of hepatic and muscle insulin substrate receptor (IRS)-1 and AKT phosphorylation. Our data show that the high fat diet caused an up-regulation of the TNF-α, IL-6, and iNOS genes. Although after intervention with yerba maté extract the expression levels of those genes returned to baseline through the NF-κB pathway, these results could also be secondary to the weight loss observed. In conclusion, our results indicate that yerba maté has a potential anti-inflammatory effect. Additionally, these data demonstrate that yerba maté inhibits hepatic and muscle TNF-α and restores hepatic insulin signalling in mice with high fat diet-induced obesity.     

Activation of the AMP-activated protein kinase enhances glucose-stimulated insulin secretion in mouse β-cells

The AMP-activated protein kinase (AMPK) is one of the key players in cellular energy regulation adapting cellular demands to nutritional and metabolic variations. Oral antidiabetic drugs like metformin and glitazones (thiazolidinediones) are known to stimulate this enzyme. Besides their established action on peripheral organs including liver and muscles, it has been claimed that these drugs may affect β-cell function. However, it is still a matter of debate whether pharmacological AMPK stimulation increases or decreases insulin secretion. To study this point and to reveal mechanisms underlying changes in insulin secretion we used the specific AMPK activator AICAR and investigated its effects on stimulus-secretion coupling. Membrane potential and currents were measured by the patch-clamp technique, [Ca (2+)]c, mitochondrial membrane potential, and NAD(P)H by fluorescence techniques and insulin secretion by a radioimmunoassay. AICAR enhanced glucose-stimulated insulin release, an effect that can be attributed to the augmentation of electrical activity and [Ca (2+)]c resulting from an AICAR-evoked inhibition of the KATP current. This latter effect was not due to a direct interaction of AICAR with the K[ATP] channels but was dependent on cell metabolism. AICAR did not affect mitochondrial membrane potential or NAD(P)H autofluorescence. Metformin mimicked the action of AICAR on electrical activity, [Ca (2+) ]c, and K[ATP] current. However, compared to AICAR the effects were less pronounced and not sufficient to stimulate insulin secretion. In conclusion, activation of AMPK augments nutrient-induced insulin secretion. Thus, targeting AMPK of β-cells may be an appropriate strategy for the treatment of disturbed glucose homeostasis..     

Matrix metalloproteinase-9 (MMP9) and high sensitivity C – Reactive protein (hs-CRP) in coronary artery ectasia

ObjectiveThe specific causative mechanisms of abnormal luminar dilatation in coronary artery ectasia (CAE) are essentially unknown. Destruction of the extracellular matrix may be responsible for ectasia formation. Thus, we investigated the role of matrix metalloproteinases (MMP9), and inflammatory marker (high-sensitive C-reactive protein) in CAE patients.MethodsThis study consisted of 30 consecutive CAE patients, 30 obstructive coronary artery disease (CAD) patients, and 20 controls with normal coronary arteries undergoing cardiac catheterization. Plasma levels of MMP-9, and hs-CRP were measured.ResultsHs-CRP level was significantly higher in the the CAE group than both in the CAD and control groups (2.3 ± 0.5, 1.19 ± 0.54, 0.8 ± 0.3 mg/l, respectively, both p < 0.001), while, MMP-9 level was significantly higher in both CAE group and CAD than control groups (27.71 ± 4.7, 25.2 ± 4.1, 18.6 ± 3.3 ng/ml, respectively , both p < 0.001). In subgroup analyses, MMP-9 level was significantly higher in CAE patients with multivessel involvement compared to those with single-vessel ectasia (29.4 ± 3.1 vs. 25.2 ± 5.5 ng/ml, P = 0.01), while hs CRP level was comparable in both groups (2.3 ± 0.52 vs. 2.4 ± 0.45 ng/ml, P = 0.82).ConclusionOur results suggest that the increased levels of MMP-9, hs-CRP may be responsible for ectasia formation in patients with CAE and plasma level of MMP-9 is correlated with the severity of CAE.     

Elevation of lung surfactant phosphatidylcholine in mouse models of Sandhoff and of Niemann–Pick A disease

Sandhoff disease is caused by the defective activity of the lysosomal enzyme beta-hexosaminidase, resulting in accumulation of the glycolipids, GA2 and GM2. Niemann-Pick A/B disease is caused by the defective activity of lysosomal acid sphingomyelinase resulting in sphingomyelin accumulation. Pulmonary complications have been observed in both diseases. We now demonstrate changes in phospholipid levels in pulmonary surfactant in mouse models of these diseases. In the Hexb mouse, a model of Sandhoff disease, lipid phosphate levels were elevated in surfactant from 3- and 4-month-old mice, which was mainly due to elevated levels of phosphatidylcholine. In the ASM mouse, a model of Niemann-Pick A disease, levels of the primary storage material, sphingomyelin, were elevated as expected, and levels of phosphatidylcholine and two other phospholipids were also significantly elevated in pulmonary surfactant and in lung tissue from 5-, 6- and 7-month-old mice. These results suggest that changes in phospholipid levels and composition in lung surfactant might be a general feature of sphingolipid storage diseases, which may be in part responsible for the increased susceptibility of these patients to respiratory infections and lung pathology, often the main reason for the death of these patients.     

Antagonising Wnt/β-catenin signalling ameliorates lens-capsulotomy-induced retinal degeneration in a mouse model of diabetes

Aims/hypothesis

Cataract surgery in diabetic individuals worsens pre-existing retinopathy and triggers the development of diabetic ocular complications, although the underlying cellular and molecular pathophysiology remains elusive. We hypothesise that lens surgery may exaggerate pre-existing retinal inflammation in diabetes, which may accelerate neurovascular degeneration in diabetic eyes.

Methods

Male heterozygous Ins2^Akita mice (3 months of age) and C57BL/6 J age-matched siblings received either lens capsulotomy (to mimic human cataract surgery) or corneal incision (sham surgery) in the right eye. At different days post surgery, inflammation in anterior/posterior ocular tissues was assessed by immunohistochemistry and proinflammatory gene expression in the retina by quantitative PCR (qPCR). Degenerative changes in the retina were evaluated by electroretinography, in vivo examination of retinal thickness (using spectral domain optical coherence tomography [SD-OCT]) and morphometric analysis of retinal neurons. The therapeutic benefit of neutralising Wnt/β-catenin signalling following lens capsulotomy was evaluated by intravitreal administration of monoclonal antibody against the co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) (Mab2F1; 5 μg/μl in each eye).

Results

Lens capsulotomy triggered the early onset of retinal neurodegeneration in Ins2^Akita mice, evidenced by abnormal scotopic a- and b-wave responses, reduced retinal thickness and degeneration of outer/inner retinal neurons. Diabetic Ins2^Akita mice also had a higher number of infiltrating ionised calcium-binding adapter molecule 1 (IBA1)/CD68⁺ cells in the anterior/posterior ocular tissues and increased retinal expression of inflammatory mediators (chemokine [C-C motif] ligand 2 [CCL2] and IL-1β). The expression of β-catenin was significantly increased in the inner nuclear layer, ganglion cells and infiltrating immune cells in Ins2^Akita mice receiving capsulotomy. Neutralisation of Wnt/β-catenin signalling by Mab2F1 ameliorated ocular inflammation and prevented capsulotomy-induced retinal degeneration in the Ins2^Akita mouse model of diabetes.

Conclusions/interpretation

Targeting the canonical Wnt/β-catenin signalling pathway may provide a novel approach for the postoperative management of diabetic individuals needing cataract surgery.

     

What kind of simple fasting index should be used to estimate insulin sensitivity in humans?

The hyperinsulinemic euglycemic glucose clamp method is the gold standard for measuring insulin resistance. However it is complex, and simple indexes have been developed. Some of them are based on formulae that calculate the product or the addition of fasting plasma insulin and glucose values whereas others are based on their ratios. We calculated several simple indexes of insulin resistance and compared them to hyperinsulinemic euglycemic clamp data in 111 subjects with a wide range of insulin resistance. We showed that indexes using insulin and glucose ratios in their formulae are poorly correlated with clamp measurements and give false evaluations, particularly in glucose-intolerant and type 2 diabetic subjects. Thus, whatever the glucose profile of study subjects, we suggest the use of a simple index based on the product or the addition of fasting plasma insulin and glucose values instead of their ratios to obtain insulin resistance evaluations close to the hyperinsulinemic euglycemic clamp technique.     

The role of insulin signaling in the development of β-cell dysfunction and diabetes

The peptide hormone insulin not only regulates metabolic pathways, but also proliferative signaling pathways. Insulin regulates cell proliferation, protein synthesis and gene expression in most, if not all, mammalian tissues. Extensive recent studies have shown that insulin also plays an important role in the regulation of pancreatic islet β-cell function. In the development of peripheral insulin resistance leading to an increased demand for insulin production, increase in β-cell mass by compensatory hyperplasia and hypertrophy of β-cells and insulin output is a crucial mechanism to maintain euglycemia. Indeed, impaired insulin signaling in the β-cells and increased β-cell apoptosis are associated with the onset of diabetes in obese insulin resistant type 2 diabetes mellitus (T2DM). Studies using gene knockout approaches in mice have further demonstrated that the insulin signaling in the β-cells is critical for mediating insulin action on them to maintain appropriate mass and insulin production. It is conceivable that insulin resistance, which is usually associated with the compensatory mechanism of hyperinsulinemia, occurring in the β-cells could be a major contributor leading to increased rate of β-cell death and declined β-cell mass. It is hypothesized that a strategy to improve intra-islet insulin action via enhancing β-cell responsiveness could be a considerable benefit in the prevention and treatment of T2DM.