Faculty Bibliography

Reports an error in "Epac2-dependent mobilization of intracellular Ca2+ by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in beta -cells of phospholipase C-epsilon knockout mice" by Igor Dzhura, Oleg G. Chepurny, Grant G. Kelley, Colin A. Leech, Michael W. Roe, Elvira Dzhura, Parisa Afshari, Sundeep Malik, Michael J. Rindler, Xin Xu, Youming Lu, Alan V. Smrcka and George G. Holz (The Journal of Physiology, 2010[Dec][15], Vol 588[24], 4871-4889). In the original article, there was an error in the Methods section entitled 'Generation of Epac2 knockout mice' on page 4873. The first sentence of that section should read 'Epac2 KO mice with global disruption of RAPGEF4 gene expression (NCBI GeneID 56508) were generated by the Texas A&M Institute for Genomic Medicine through customized service for Dr. Lu at Louisiana State University Health Sciences Center'. (The following abstract of the original article appeared in record 2011-11969-007). Calcium can be mobilized in pancreatic beta -cells via a mechanism of Ca2+-induced Ca2+ release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in beta -cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C-epsilon (PLC-epsilon ) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC-epsilon gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the beta -cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse beta -cells were loaded with the photolabile Ca2+ chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca2+ generated CICR in only 9% of the beta -cells tested, whereas CICR was generated in 82% of the beta -cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A(6-Bnz-cAMP-AM)orEpac2 (8-pCPT-2'-O-Me-cAMP-AM)selectively. However, in beta -cells of PLC-epsilon KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC-epsilon KO beta -cells with recombinant PLC-epsilon rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC-epsilon with amutated Ras association (RA) domain, or a H1640L PLC-epsilon that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT beta -cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC-epsilon exists in beta -cells, and that the activities of Epac2 and PLC-epsilon are key determinants of CICR in this cell type.

Background: We previously reported human catalytic autoantibodies to amyloid b peptide (Ab). We hypothesize that recognition of electrophilic amyloid epitopes by nucleophilic autoantibodies is an innate immune function that is recruited for catalytic clearance of amyloid deposits associated with aging and Alzheimer's disease (AD). Methods: Ab cleavage was measured by HPLC, acid precipitation, mass spectroscopy or electrophoresis. Electrophilic Ab (E-Ab) was prepared by carbonylation with the lipid peroxidation end products 4-hydroxynonenal (HNE)/malonaldehyde (MDA) or phosphonate diester insertion. Covalent immune complexes were analyzed by SDS-electrophoresis. Ab1-42 aggregates were identified by antibody or Thioflavin-T staining. Results: IgM from healthy human sera, the first antibody class produced during B cell differentiation, catalyzed Ab cleavage at rates superior to IgGs. Preferential Ab cleavage by IgMs was also observed for antibodies from the sera and cerebrospinal fluid from patients with AD. Two Ab cleaving antibody fragments were isolated from a phage library, a heterodimeric V L -V L construct (2E6) and a single domain V L construct (5D3). Treatment with antibody 2E6 induced disappearance of oligomeric and fibrillar Ab. Intracranial antibody injection in Ab-overexpressing transgenic mice cleared the Ab plaques. Traditional antibodies bind antigens at complementarity determining regions (CDRs). The Ab cleaving antibodies contained CDRs with no or minimum mutations acquired by antigen-driven diversification. Deleting the CDRs did not attenuate Ab cleavage by antibody 2E6 but the catalytic activity was lost by replacing the framework regions (FRs) with corresponding FRs from a non-catalytic antibody. The FRs are evolutionarily conserved segments important for innate recognition of B cell superantigens without requirement for adaptive immune processes. From protease inhibitor and epitope mapping studies, the catalytic mechanism entails noncovalent binding at the Ab C terminus followed by nucleophilic peptide bond cleavage. Antibody 2E6 reacted covalently with an electrophilic phosphonate-containing Ab analog and the naturally-occurring Ab-HNE/Ab-MDA analogs (E-Ab). Monoclonal murine antibodies (MAbs) that cleaved Ab at low substrate concentrations were identified by immunization with non-electrophilic Ab. A subset of MAbs induced by immunization with E-Ab cleaved Ab robustly. Conclusions: Amplification of the innate noncovalent recognition and catalytic functions of antibodies driven by age/ disease-associated Ab accumulation can remove toxic amyloid deposits

Transgenic mice are used increasingly to model brain amyloidosis, mimicking the pathogenic processes involved in Alzheimer's disease (AD). In this chapter, an in vivo strategy is described that has been successfully used to map amyloid-beta deposits in transgenic mouse models of AD with magnetic resonance imaging (MRI), utilizing both the endogenous contrast induced by the plaques attributed to their iron content and by selectively enhancing the signal from amyloid-beta plaques using molecular-targeting vectors labeled with MRI contrast agents. To obtain sufficient spatial resolution for effective and sensitive mouse brain imaging, magnetic fields of 7-Tesla (T) or more are required. These are higher than the 1.5-T field strength routinely used for human brain imaging. The higher magnetic fields affect contrast agent efficiency and dictate the choice of pulse sequence parameters for in vivo MRI, all addressed in this chapter. Two-dimensional (2D) multi-slice and three-dimensional (3D) MRI acquisitions are described and their advantages and limitations are discussed. The experimental setup required for mouse brain imaging is explained in detail, including anesthesia, immobilization of the mouse's head to reduce motion artifacts, and anatomical landmarks to use for the slice alignment procedure to improve image co-registration during longitudinal studies and for subsequent matching of MRI with histology.

A long standing problem with antidepressant drugs is their delayed onsets of action which has made them unsatisfactory in the rapid treatment of serious depressions involving agitated and suicidal behavior. Two new approaches to this problem involve the glutamatergic antagonist, ketamine, recently reviewed, and the acute inhibition of central stress circuits, which is the subject of the present review. The rationale behind stress-circuit inhibition comes from the findings that both clinical and experimental depressions are accompanied by increased neural activity in the stress network together with inhibited activity in regions underlying active motivated behaviors, and that both changes are reversed by effective antidepressant treatment. It has been shown further that direct pharmacological inhibition of central noradrenergic stress nuclei produces immediate reversal of depressive-like passive as well as sickness behaviors. Dipivalyl-6-fluoronorepinephrine (dp6FNE), a brain-permeable pro-drug of 6FNE, a full agonist at inhibitory brain alpha- adrenoceptors in brainstem noradrenergic stress nuclei, has been developed and found in preliminary studies to cause rapid reversal of both passivity and anhedonia as well as anxiolysis without significant hypoactivity in the open field. Low doses of agonists of glucocorticoid and 5HT1A autoreceptors, which respectively inhibit the hypothalamic-pituitaryadrenal axis and serotonergic stress systems, may also share these effects as well as potentiate the actions of dp6FNE. Anti-stress effects may also be involved in the rapid therapeutic effects found with intracerebral administration of first generation antidepressants and may prove helpful in potentiating the therapeutic actions of stimulants. Stone et al

We report the elusive X-ray structure of the Dess-Martin periodinane (DMP), a hypervalent iodine reagent popular amongst synthetic chemists. In the solid state, the highly crystalline compound forms an intricate coordination polymer held together by intermolecular halogen and hydrogen bonds.

Tauopathy in the brain of patients with Alzheimer's disease starts in the entorhinal cortex (EC) and spreads anatomically in a defined pattern. To test whether pathology initiating in the EC spreads through the brain along synaptically connected circuits, we have generated a transgenic mouse model that differentially expresses pathological human tau in the EC and we have examined the distribution of tau pathology at different timepoints. In relatively young mice (10-11 months old), human tau was present in some cell bodies, but it was mostly observed in axons within the superficial layers of the medial and lateral EC, and at the terminal zones of the perforant pathway. In old mice (>22 months old), intense human tau immunoreactivity was readily detected not only in neurons in the superficial layers of the EC, but also in the subiculum, a substantial number of hippocampal pyramidal neurons especially in CA1, and in dentate gyrus granule cells. Scattered immunoreactive neurons were also seen in the deeper layers of the EC and in perirhinal and secondary somatosensory cortex. Immunoreactivity with the conformation-specific tau antibody MC1 correlated with the accumulation of argyrophilic material seen in old, but not young mice. In old mice, axonal human tau immunoreactivity, especially at the endzones of the perforant pathway, was greatly reduced. Relocalization of tau from axons to somatodendritic compartments and propagation of tauopathy to regions outside of the EC correlated with mature tangle formation in neurons in the EC as revealed by thioflavin-S staining. Our data demonstrate propagation of pathology from the EC and support a trans-synaptic mechanism of spread along anatomically connected networks, between connected and vulnerable neurons. In general, the mouse recapitulates the tauopathy that defines the early stages of AD and provides a model for testing mechanisms and functional outcomes associated with disease progression.

BACKGROUND: Mutations in any of the five subunits of eukaryotic translation initiation factor 2B (eIF2B) can lead to an inherited chronic-progressive fatal brain disease of unknown aetiology termed leucoencephalopathy with vanishing white matter (VWM). VWM is one of the most prevalent childhood white matter disorders, which markedly deteriorates after inflammation or exposure to other stressors. eIF2B is a major housekeeping complex that governs the rate of global protein synthesis under normal and stress conditions. A previous study demonstrated that Eif2b5(R132H/R132H) mice suffer delayed white matter development and fail to recover from cuprizone-induced demyelination, although eIF2B enzymatic activity in the mutant brain is reduced by merely 20%. PRINCIPAL FINDINGS: Poor astrogliosis was observed in Eif2b5(R132H/R132H) mice brain in response to systemic stress induced by peripheral injections of lipopolysaccharide (LPS). Even with normal rates of protein synthesis under normal conditions, primary astrocytes and microglia isolated from mutant brains fail to adequately synthesise and secrete cytokines in response to LPS treatment despite proper induction of cytokine mRNAs. CONCLUSIONS: The mild reduction in eIF2B activity prevents the appropriate increase in translation rates upon exposure to the inflammatory stressor LPS. The data underscore the importance of fully-functional translation machinery for efficient cerebral inflammatory response upon insults. It highlights the magnitude of proficient translation rates in restoration of brain homeostasis via microglia-astrocyte crosstalk. This study is the first to suggest the involvement of microglia in the pathology of VWM disease. Importantly, it rationalises the deterioration of clinical symptoms upon exposure of VWM patients to physiological stressors and provides possible explanation for their high phenotypic variability.