Mechanisms of neuroprotection against ischemic insult by stress-inducible phosphoprotein-1/prion protein complex.

Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrPC ) and activating α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1-h oxygen-glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrPC . Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrPC and α7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrPC /STI1 engagement and its subsequent downstream signaling cascades involving α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults.

Susceptibility tensor imaging of the kidney and its microstructural underpinnings.

PURPOSE: The purpose of this study was to determine whether susceptibility tensor imaging (STI) could overcome limitations of current techniques to detect tubules throughout the kidney. METHODS: Normal mouse kidneys (n = 4) were imaged at 9.4T using a three-dimensional gradient multi-echo sequence (55-micron isotropic resolution). Phase images from 12 orientations were obtained to compute the susceptibility tensor. Diffusion tensor imaging (DTI) with 12 encoding directions was compared with STI. Tractography was performed to visualize and track the course of tubules with DTI and STI. Confocal microscopy was used to identify which tubular segments of the nephron were detected by DTI and STI. RESULTS: Diffusion anisotropy was limited to the inner medulla of the kidney. DTI did not find a significant number of coherent tubular tracks in the outer medulla or cortex. With STI, we found strong susceptibility anisotropy and many tracks in the inner and outer medulla and in limited areas of the cortex. CONCLUSION: STI was able to track tubules throughout the kidney, whereas DTI was limited to the inner medulla. STI provides a novel contrast mechanism related to local tubule microstructure and may offer a powerful method to study the nephron.

Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.

The dopamine transporter (DAT) plays a key role in the regulation of dopaminergic signaling wherein it controls both the spatial and temporal actions of dopamine. Here we evaluated the behavioral and neurochemical consequences of increased DAT function by generating DAT transgenic mice (DAT-tg) that overexpress the transporter. These mice were generated by pronuclear injection of a bacterial artificial chromosome containing the mouse DAT locus, yielding an anatomical expression pattern of DAT-tg identical to WT. In DAT-tg mice there is a 3-fold increase in the levels of total and membrane-expressed DAT, but synaptic plasma membrane fractions of DAT-tg mice show only a 30% increase in transporter levels. Functional studies reveal that in the DAT-tg animals there is a 50% increase in the rate of dopamine (DA) uptake resulting in extracellular levels of DA that are decreased by approximately 40%. Behaviorally, DAT-tg animals display similar locomotor stimulation when treated with DAT blockers such as GBR12909, methylphenidate, and cocaine. However, these mice demonstrate markedly increased locomotor responses to amphetamine compared with WT animals. Furthermore, compared with controls, there is a 3-fold greater increase in the amount of DA released by amphetamine in DAT-tg mice that correlates with the 3-fold increase in protein expression. Finally, DAT-tg animals show reduced operant responding for natural reward while displaying preference for amphetamine at much lower doses (0.2 and 0.5 mg/kg) than WT mice (2 mg/kg). These results suggest that overexpression of DAT leads to a marked increase in sensitivity to psychomotor and rewarding properties of amphetamine.

Slow-release delivery enhances the pharmacological properties of oral 5-hydroxytryptophan: mouse proof-of-concept.

5-hydroxytryptophan (5-HTP) has shown therapeutic promise in a range of human CNS disorders. But native 5-HTP immediate release (IR) is poorly druggable, as rapid absorption causes rapid onset of adverse events, and rapid elimination causes fluctuating exposure. Recently, we reported that 5-HTP delivered as slow-release (SR) in mice augmented the brain pro-serotonergic effect of selective serotonin reuptake inhibitors (SSRIs), without the usual adverse events associated with 5-HTP IR. However, our previous study entailed translational limitations, in terms of route, dose, and duration. Here we modeled oral 5-HTP SR in mice by administering 5-HTP via the food. We modeled oral SSRI treatment via fluoxetine in the water, in a regimen recapitulating clinical pharmacokinetics and pharmacodynamics. 5-HTP SR produced plasma 5-HTP levels well within the range enhancing brain 5-HT function in humans. 5-HTP SR robustly increased brain 5-HT synthesis and levels. When administered with an SSRI, 5-HTP SR enhanced 5-HT-sensitive behaviors and neurotrophic mRNA expression. 5-HTP SR's pro-serotonergic effects were stronger in mice with endogenous brain 5-HT deficiency. In a comprehensive screen, 5-HTP SR was devoid of overt toxicological effects. The present preclinical data, appreciated in the context of published 5-HTP clinical data, suggest that 5-HTP SR could represent a new therapeutic approach to the plethora of CNS disorders potentially treatable with a pro-serotonergic drug. 5-HTP SR might in particular be therapeutically relevant when brain 5-HT deficiency is pathogenic and as an adjunctive augmentation therapy to SSRI therapy.

Alternate signalling pathways from the interleukin-2 receptor.

Interleukin-2 (IL-2) plays a major role in the proliferation of cell populations during an immune reaction. The beta(c) and gamma(c) subunits of the IL-2 receptor (IL-2R) are sufficient and necessary for signal transduction. Despite lacking known catalytic domains, receptor engagement leads to the activation of a diverse array protein tyrosine kinases (PTKs). In resting or anergised T cells, Jak3 is not activated. Signals arising from the PROX domain of the gamma(c) subunit activate p56(lck) (lck) leading to the induction of anti-apoptotic mechanisms. When Jak3 is activated, in primed T cells, other PTKs predominantly mediate the induction of anti-apoptotic mechanisms and drive cellular proliferation. This review intends to suggest a role for these differences within the context of the immune system.

Expression of an anti-CD33 single-chain antibody by Pichia pastoris.

CD33 is a cell surface glycoprotein expressed on cells of myelomonocytic lineage, leukaemic cells, but not haematopoietic stem cells. By virtue of its expression pattern, CD33 has become a popular target for new immunotherapeutic approaches to treat acute myeloid leukaemia. The methylotrophic yeast Pichia pastoris strain KM71H was used to produce an anti-CD33 single chain variable fragment (scFv), with the intention of conjugation to a radioisotope, for therapeutic use. To direct secreted expression of the anti-CD33-scFv the alpha-mating factor secretory signal sequence (alpha-MF) was used, with constructs containing a complete (CS) and incomplete (INCS) cleavage site to accommodate the potential outcomes of dibasic endopeptidase, Kex2, and dipeptidyl amino peptidase, Ste13, processing. The anti-CD33-scFv was expressed in BMMY cultures using both constructs, with a final yield of 48 mg/l (CS) and 11 mg/l (INCS). N-terminal sequencing showed that the CS-scFv had not been cleaved by Ste13, leaving amino acids EAEA at the N-terminus. The INCS-scFv construct produced a mixture of 50% authentic scFv and 50% with 11 amino acids from the alpha-MF remaining at the N-terminus. Despite the aberrations in alpha-MF processing, the anti-CD33-scFv's produced from both constructs were found to be functional. Flow cytometry and Biacore analysis demonstrated binding to target antigen CD33 on the surface of human leukaemic cell line HL-60, and to recombinant soluble CD33 respectively.

Integrated approaches to understanding antipsychotic drug action at GPCRs.

The G-protein coupled receptor (GPCR) family of genes represents one of the largest druggable families of genes in the human genome. This is evident by the fact that approximately 30% of currently marketed drugs target GPCRs. However, many of these drugs are limited in their clinical potential as they are associated with debilitating side effects-a consequence of our incomplete understanding of their pharmacology and the signaling pathways regulated by GPCRs. Because of the limited range of tools available to resolve these issues, integrated approaches are required to fully understand the pharmacological action of drugs and the biochemical repertoire regulated by GPCRs. In this review we will focus on the action of antipsychotic drugs on certain monoamine GPCRs in the central nervous system (CNS) and the approaches being developed to elucidate their distinct pharmacology.

Neuroanatomical phenotypes in the reeler mouse.

The reeler mouse (Reln) has been proposed as a neurodevelopmental model for certain neurological and psychiatric conditions and has been studied by qualitative histochemistry and electron microscopy. Using magnetic resonance microscopy (MRM), we have quantitated for the first time the neuromorphology of Reln mice at a resolution of 21.5 microm. The neuroanatomical phenotypes of heterozygous and homozygous mutant Reln mice were compared to those of wild type (WT) littermates using morphometry and texture analysis. The cortical, hippocampal, and cerebellar phenotypes of the heterozygous and homozygous mutant Reln mice were confirmed, and new features were revealed. The Reln(rl/rl) mice possessed a smaller brain, and both Reln(rl/+) and Reln(rl/rl) mice had increased ventricles compared to WT controls. Shape differences were found between WT and Reln(rl/rl) brains, specifically in cerebellum, olfactory bulbs, dorsomedial frontal and parietal cortex, certain regions of temporal and occipital lobes, as well as in the lateral ventricles and ventral hippocampus. These findings suggest that certain brain regions may be more severely impacted by the Reln mutation than others. Gadolinium-based active staining demonstrated that layers of the hippocampus were disorganized in Reln(rl/rl) mice and differences in thickness of these layers were identified between WT and Reln(rl/rl) mice. The intensity distributions characteristic to the dorsal, middle, and ventral hippocampus were altered in the Reln(rl/rl), especially in the ventral hippocampus. These differences were quantified using skewness and modeling the intensity distributions with a Gaussian mixture. Our results suggest that structural features of Reln(rl/rl) brain most closely phenocopy those of patients with Norman-Roberts lissencephaly.

Trauma and posttraumatic stress in users of the Anxiety Disorders Association of America Web site.

The Web site for the Anxiety Disorders Association of America (ADAA) receives more than 5 million visits per month and thus represents a unique medium for the study of anxiety disorders. ADAA Web site users from October 2002 to January 2003 were invited to complete a survey oriented toward trauma history and psychiatric sequelae. A diagnostic approximation of posttraumatic stress disorder (PTSD) was based on responses to the Trauma Questionnaire, the Davidson Trauma Scale, and questions about impairment. The Connor-Davidson Resilience Scale was also used. Variables were tested for their association with PTSD. Among 1558 participants, 87% had a history of trauma, and 38% had current PTSD. The population was comprised predominantly of white middle-class women, half of whom were married. More than 90% were first-time users of the site. Factors associated with PTSD included death of, or harm to, a loved one; personal history of incest, rape, or physical abuse; lower age; lower income; unemployment; missed work; increased medical care; dissatisfaction with psychotropic medication; depressive symptoms; and lower resilience. In this selective convenience sample, there were high rates of traumatization and PTSD. The demographics of this group are similar to those seen in previously studied populations that had contacted the ADAA. Furthermore, the factors associated with PTSD were like those in many community surveys. The ADAA Web site has the opportunity to benefit large numbers of highly distressed individuals.

Possible mechanism for the alpha subunit of the interleukin-2 receptor (CD25) to influence interleukin-2 receptor signal transduction.

The receptors for interleukin 2 (IL-2) and interleukin 15 (IL-15) in T cells share the IL-2R beta subunit (CD122) and gamma(C) subunit but have private alpha subunits. Despite utilizing the same receptor chains known to be necessary and sufficient to transduce IL-2 signals the two cytokines manifest different cellular effects. It is commonly held that the alpha subunit of the IL-2R (CD25) is involved solely in the generation of a high affinity receptor complex. This is questioned by the development of autoimmune diseases in instances where the expression of CD25 is absent. The timely expression of CD25 in the thymus has been linked with clonal deletion. Evidence from peripheral T cells indicates that survival signals arising from the intermediate affinity IL-2R (lacking CD25) do not require the activation of Janus kinase 3 (Jak3) but do require the presence of the membrane proximal region of the gamma(C) chain. This particular signalling pathway is not observed in the high affinity receptor complex where Jak3 is activated. Recent data point to CD25 having a surface distribution consistent with it being localized within membrane microdomains. Here we suggest that in the absence of CD25 expression, IL-2R activation occurs within the soluble membrane fraction. This membrane environment and the absence of CD25 promotes Jak3 independent signal transduction and induction of antiapoptotic mechanisms. T cell antigen receptor (TCR) signalling leads to the induction of CD25 expression, which localizes to membrane microdomains. There is a dynamic pre-association of CD25 and CD122 leading to the loose association of the heterodimer with membrane microdomains. High affinity IL-2R signalling in the context of CD25 and the microdomain environment is characterized by Jak3 activation. The relative levels of high to intermediate affinity receptor signalling determines whether a cell proliferates or undergoes activation induced cell death dependent upon cell status.