Selective Defect Research Articles

PS18 - 87. A novel FPLD-associated PPARgamma mutant (E379K) displays a selective defect in target gene transcription

Familial partial lipodystrophy type 3 (FPLD) is an autosomal dominant disease characterized by aberrant adipose distribution and metabolic disturbances, including insulin resistance and dyslipidemia.

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) α and β loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders(1-4). T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the αβTCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the αβTCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC(5). Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events(6). For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes(7-9). Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCRα chain at the DN stage, resulting in premature negative selection. Our lab has developed the HY(cd4) model, in which the transgenic HY TCRα is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice(10). Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HY(cd4) mouse model. While negative selection in HY(cd4) mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

Development of targeted biologics for systemic lupus erythematosus

The prototypic autoimmune disease, systemic lupus erythematosus (SLE), is known to be associated with a number of genetic polymorphisms that can affect B cell function as well as polyclonal B cell hyperactivity. Developing an understanding of the complex nature of human B cell activation and differentiation has permitted an assessment of whether specific stages of B cell maturation are affected by the tendency for polyclonal B cell activation. Moreover, the analysis of perturbations of the specific stages of B cell maturation has generated new information on whether abnormalities in B cell differentiation are primarily involved in SLE immunopathology or, rather, are secondary to the inflammatory environment characteristic of subjects with this autoimmune disease. Multivariant flow cytometric analysis has documented abnormalities in B cell maturation that are primarily associated with lupus, or, alternatively related to disease duration, disease activity and concomitant medication. Together, these analyses have provided new insights on the role of B cell over-reactivity in SLE. Characterization of peripheral blood B cell subsets in patients with SLE has provided unique opportunities to identify abnormalities among pre-naive, transitional, pre-naive, memory B cells and in particular plasmablasts/plasma cells including indications for defects in negative selection of autoreactive B cells at certain stages. The pathogenic impact of all of these individual disturbances remains less clear, although the findings have served to identify SLE as associated with numerous abnormalities in B cell development. Moreover, active SLE is characterized by overactive T cell dependent germinal center-like reactions that produce expanded and largely unregulated numbers of memory B cells and plasma cells, thereby contributing to SLE pathogenesis. Finally, an understanding of the nature of the B cell abnormalities in SLE has generated new targets for therapy of this disease. The first of these, belimumab, a monoclonal antibody to the B cell survival factor B Lymphocyte stimulator (BLyS), has proven successful in treating patients with SLE. Additional approaches that target specific stages of B cell activation or differentiation are in development for treatment of SLE.

Genetic interactions of hypomorphic mutations in the m7G cap-binding pocket of yeast nuclear cap binding complex: an essential role for Cbc2 in meiosis via splicing of MER3 pre-mRNA.

Nuclear cap binding protein complex (CBC) is a heterodimer of a small subunit (Cbc2 in yeast) that binds the m(7)G cap and a large subunit (Sto1 in yeast) that interacts with karyopherins. In order to probe the role of cap recognition in yeast CBC function, we introduced alanine mutations (Y24A, F91A, D120A, D122A, R129A, and R133A) and N-terminal deletions (NΔ21 and NΔ42) in the cap-binding pocket of Cbc2. These lesions had no effect on vegetative growth, but they ameliorated the cold-sensitivity of tgs1Δ cells that lack trimethylguanosine caps (a phenotype attributed to ectopic association of CBC with the m(7)G cap of the normally TMG-capped U1 snRNA), thereby attesting to their impact on cap binding in vivo. Further studies of the Cbc2-Y24A variant revealed synthetic lethality or sickness with null mutations of proteins involved in early steps of spliceosome assembly (Nam8, Mud1, Swt21, Mud2, Ist3, and Brr1) and with otherwise benign mutations of Msl5, the essential branchpoint binding protein. Whereas the effects of weakening CBC-cap interactions are buffered by other actors in the splicing pathway during mitotic growth, the NΔ42 allele causes a severe impediment to yeast sporulation and meiosis. RNA analysis revealed a selective defect in the splicing of MER3 and SAE3 transcripts in cbc2-NΔ42 diploids during attempted sporulation. An intronless MER3 cDNA fully restored sporulation and spore viability in the cbc2-NΔ42 strain, signifying that MER3 splicing is a limiting transaction. These studies reveal a new level of splicing control during meiosis that is governed by nuclear CBC.

Rod-Driven OFF Pathway Responses in the Distal Retina: Dark-Adapted Flicker Electroretinogram in Mouse

PurposeThe rodent retina does not exhibit a positive OFF-response in the electroretinogram (ERG), which makes it difficult to evaluate its OFF-pathway functions in vivo. We studied the rod-driven OFF pathway responses by using a dark-adapted 10-Hz flicker ERG procedure in mouse.Materials and MethodsConventional ERGs and 10-Hz dark-adapted flicker ERGs were obtained in wild-type mice (C57BL/6), in mice with pure rod (cpfl1) or pure cone (rho−/−) function, and in nob1 mice which have a selective ON-pathway defect. To isolate the response from ON or OFF pathway, glutamate analogs 2-amino-4-phosphobutyric acid (APB, an ON pathway blocker) and cis-2, 3-piperidine-dicarboxylic acid (PDA, an OFF pathway blocker), were injected intravitreally.ResultsThe amplitude-intensity profile of the dark-adapted 10-Hz flicker ERG in the wild-type mice exhibits two peaks at middle and high light intensities. The two peaks represent rod- and cone-driven responses respectively. In APB-treated C57BL/6 mice and in nob1 mice, the dark-adapted ERG b-waves were absent. However, both rod- and cone-driven OFF pathway responses were evident with flicker ERG recording. At middle light intensities that activate only rod system, the flicker ERG responses in saline-injected nob1 mice were similar to those in APB-injected cpfl1 mice and wild-type mice. These responses are sensitive to PDA. The amplitudes of these rod-driven OFF pathway responses were approximately 20% of the total rod-driven flicker ERG responses.ConclusionWe demonstrate that the rod-OFF bipolar cell pathway is functional in the outer retina. The dark-adapted flicker ERG is practical for the evaluation of rod- and cone-driven responses, and the residual OFF pathway signals in subjects with ON pathway defects.

Upregulation of Retinal Dehydrogenase 2 in Alternatively Activated Macrophages during Retinoid-dependent Type-2 Immunity to Helminth Infection in Mice

Although the vitamin A metabolite retinoic acid (RA) plays a critical role in immune function, RA synthesis during infection is poorly understood. Here, we show that retinal dehydrogenases (Raldh), required for the synthesis of RA, are induced during a retinoid-dependent type-2 immune response elicited by Schistosoma mansoni infection, but not during a retinoid-independent anti-viral immune response. Vitamin A deficient mice have a selective defect in TH2 responses to S. mansoni, but retained normal LCMV specific TH1 responses. A combination of in situ imaging, intra-vital imaging, and sort purification revealed that alternatively activated macrophages (AAMφ) express high levels of Raldh2 during S. mansoni infection. IL-4 induces Raldh2 expression in bone marrow-derived macrophages in vitro and peritoneal macrophages in vivo. Finally, in vivo derived AAMφ have an enhanced capacity to induce Foxp3 expression in CD4+ cells through an RA dependent mechanism, especially in combination with TGF-β. The regulation of Raldh enzymes during infection is pathogen specific and reflects differential requirements for RA during effector responses. Specifically, AAMφ are an inducible source of RA synthesis during helminth infections and TH2 responses that may be important in regulating immune responses.

Improving outcomes for composite grafts in nasal reconstruction

Auricular composite grafting is a useful technique for the reconstruction of select nasal defects. However, unpredictable graft survival has been the primary limitation of this technique. The literature was reviewed to evaluate the effectiveness of various surgical, pharmacologic, hyperbaric, and hypothermic interventions to improve composite graft survival. Although no statistically significant surgical interventions were found, several techniques appear promising. There are a number of studies demonstrating the beneficial effects of perioperative and postoperative corticosteroid administration. Other agents studied have had modest to no benefit. Hyperbaric oxygen therapy holds promise but the expense, practicality, and lack of an established optimal treatment protocol remain obstacles to routine use. The use of corticosteroids and postoperative cooling are relatively inexpensive and effective modalities to improve auricular composite graft survival when used for nasal reconstruction. Hyperbaric oxygen therapy may have a role, but more research is needed before it is employed routinely.

Reduced severity of peanut-induced anaphylaxis in TLR9-deficient mice is associated with selective defects in humoral immunity.

Signaling through the innate immune system can promote or suppress allergic sensitization. TLR9 has modulatory effects on the mucosal immune system, and we hypothesized that TLR9 would influence susceptibility to allergic sensitization to foods. We observed that TLR9−/− mice were resistant to peanut-induced anaphylaxis. This was associated with a significant impairment in total IgE and peanut-specific IgE and IgA, but not IgG1 or Th2 cytokine production. TLR9−/− mice had reduced development of Peyer’s patches, but resistance to sensitization was not restricted to oral routes. Rag1-deficient mice were reconstituted with TLR9+/+ or −/− B cells plus CD4+ T cells. TLR9−/− B cells regained the ability to produce IgE in the presence of a wild-type environment. Our results demonstrate that TLR9 on an unknown cell type is required for the development of IgE-producing B cells, and we conclude that TLR9 signaling indirectly shapes the immune response for optimal IgE production.

Translating PI3K-Delta Inhibitors to the Clinic in Chronic Lymphocytic Leukemia: The Story of CAL-101 (GS1101)

Targeted therapy with imatinib has transformed the treatment of chronic myeloid leukemia (CML). Unlike CML, chronic lymphocytic leukemia (CLL) lacks a common genetic aberration but does demonstrate constitutive activation of PI3-kinase (PI3K) as compared to normal B cells. This constitutively active PI3K in CLL likely relates to tonic B-cell receptor signaling that is present across a wide variety of B-cell malignancies. Although PI3K is quite proximal and represents an ideal target to pharmacologically modulate, the complexity of this pathway on which many normal functions are dependent had for many years been problematic. The p110 delta isoform of PI3K is relatively specific to hematopoietic cells, and elegant mouse studies where p110 delta was genetically inactivated demonstrated only a selective B-cell defect. Subsequent development of a potent, selective p110 delta inhibitor prompted translation into the clinic for the treatment of CLL and low-grade non-Hodgkin lymphoma (NHL). From the first patient treated where a dramatic early nodal response was noted, considerable excitement has developed for this class of drugs in CLL and NHL. We will summarize the development process of CAL-101 (now GS1101) in the treatment of chronic lymphoid malignancies such as CLL.

PMO-141 Flagellin-induced IL-6 production is selectively impaired in patients with cirrhosis

IntroductionThe innate immune response is an important determinant of progression in chronic inflammatory liver diseases such as alcoholic (ALD) and non-alcoholic fatty liver disease (NAFLD). Sepsis is a frequent cause...

Nuclear factor of activated T cells (NFATc4) is required for BDNF-dependent survival of adult-born neurons and spatial memory formation in the hippocampus

New neurons generated in the adult dentate gyrus are constantly integrated into the hippocampal circuitry and activated during encoding and recall of new memories. Despite identification of extracellular signals that regulate survival and integration of adult-born neurons such as neurotrophins and neurotransmitters, the nature of the intracellular modulators required to transduce those signals remains elusive. Here, we provide evidence of the expression and transcriptional activity of nuclear factor of activated T cell c4 (NFATc4) in hippocampal progenitor cells. We show that NFATc4 calcineurin-dependent activity is required selectively for survival of adult-born neurons in response to BDNF signaling. Indeed, cyclosporin A injection and stereotaxic delivery of the BDNF scavenger TrkB-Fc in the mouse dentate gyrus reduce the survival of hippocampal adult-born neurons in wild-type but not in NFATc4(-/-) mice and do not affect the net rate of neural precursor proliferation and their fate commitment. Furthermore, associated with the reduced survival of adult-born neurons, the absence of NFATc4 leads to selective defects in LTP and in the encoding of hippocampal-dependent spatial memories. Thus, our data demonstrate that NFATc4 is essential in the regulation of adult hippocampal neurogenesis and identify NFATc4 as a central player of BDNF-driven prosurvival signaling in hippocampal adult-born neurons.

SLP-2 regulates T cell activation by optimizing cardiolipin compartmentalization in mitochondrial membranes and enhancing cellular respiration (178.7)

Abstract Stomatin-like protein 2 (SLP-2) is a mitochondrial protein of the highly conserved Stomatin, Prohibitin, Flotillin, HflC/K superfamily. The function of SLP-2 has remained unclear but members of this superfamily have been linked to membrane organization. We have previously reported that SLP-2 partitions within glycolipid-enriched, detergent-insoluble microdomains, and its expression is up-regulated upon T cell activation. This correlates with increased mitochondrial biogenesis and function. Using human inducible over-expression/down-regulation systems and newly generated T cell-specific SLP-2 knockout mice, we now report that SLP-2 binds directly and selectively to cardiolipin (CL) and interacts with prohibitins. Overexpression of SLP-2 leads to increased recruitment of prohibitins to mitochondrial membranes. This translates into optimal assembly and function of complexes I and II+III of the respiratory chain. SLP-2 deficiency in T cells is associated with abnormal CL compartmentalization and decreased respiration, which correlates with a selective post-transcriptional defect in the production of IL-2 and defective T cell (mostly CD4+) responses. Therefore, we propose that SLP-2 functions as an organizer of cardiolipin-enriched domains in mitochondrial membranes. These domains are required for optimal respiration and metabolic capacity during T cell activation. Our work also reveals a novel post-transcriptional checkpoint for IL-2 production under metabolic control.

Enumeration and characterization of self-reactive T cells in polyclonal populations (111.25)

Abstract The thymic processes of positive and negative selection are crucial for the formation of a functional T cell repertoire. TCR transgenic models have provided crucial insight into these processes, but it has not been possible to quantitate positive and negative selection in normal diverse polyclonal populations. We developed a Nur77GFP transgenic reporter mouse that specifically expresses GFP in T cells in response to TCR stimulation. Using Nur77GFP mice crossed to mice deficient in the pro-apoptotic molecule Bim, we found that two times more DP thymocytes are negatively selected by high affinity interactions than are positively selected by low affinity interactions. We also identified the fraction of SP thymocytes undergoing further deletion in the medulla, after positive selection. Cells that are rescued from negative selection have increased Nur77GFP compared to foxP3+ T regulatory cells, suggesting that T cells undergoing negative selection have TCRs with higher affinity for self compared to developing T regulatory cells. Finally, our results identify a population of self-reactive CD25-CD44+ CD4 T cells in mesenteric LNs, which have a phenotype consistent with clonal anergy. Future studies using this model will allow us to identify and quantitate selection defects in various mutant or autoimmune prone animals.

Diastolic dysfunction following HIV infection

The development of myocardial dysfunction in patients following HIV infection continues to be widely reported [1–6]. Compelling evidence of surprisingly frequent diastolic dysfunction followed by systolic dysfunction is observed in both the pediatric and adult populations [1–6]. Basic mechanisms proposed to explain these clinical observations include direct effects of HIV proteins, or indirect effects of cytokines, co-infection, autoimmunity and antiretroviral toxicity [1,7]. The advent of highly active antiretroviral therapies (HAART) has prolonged survival, but has been considered a potential source of cardiovascular complications of HIV infection [1]. Recent clinical studies in countries lacking universal access to HAART have provided some insights into the relative importance of HIV infection as opposed to its pharmacotherapy in HIV cardiomyopathy. These recent studies indicate that HIV cardiomyopathy is more prevalent among patients with HIV alone compared with those patients with HIV who also received HAART [1–6]. Thus, HIV infection appears to contribute more to HIV cardiomyopathy than does its treatment. Human autopsy data identifying gp120 in the hearts of HIV cardiomyopathy patients prior to HAART also support its potential pathogenic role in this condition [8]. The study by Kelly et al.[9] in this issue of AIDS provides an exciting and timely addition to the literature supporting a pathogenic role for HIV proteins in myocardial dysfunction following HIV infection. The authors use the most elegant and definitive hemodynamic and echocardiographic techniques to convincingly demonstrate the presence of diastolic dysfunction in their simian immunodeficiency virus (SIV) model. Their data are quite consistent with diastolic dysfunction in HIV patients that has been reported using echocardiographic methods. The authors found up-regulation of macrophage markers in the myocardium of SIV-infected animals that did not correlate with functional decline. On the contrary, SIV-induced diastolic dysfunction was strongly correlated with the extent of SIV replication in the myocardium. The authors concluded that their data support a pathogenic role for HIV virus or viral proteins in myocardial dysfunction. These clinical reports stimulated efforts to study the direct effects of recombinant HIV proteins in animal models to appreciate their potential pathogenic role in HIV cardiomyopathy [10–15]. The HIV coat protein, gp120, was reported to enhance IL-1beta-induced inducible nitric oxide synthase expression and nitric oxide production in neonatal rat cardiac myocytes in vitro[10]. A direct, negative inotropic effect of HIV gp120 on adult rat ventricular myocytes (ARVMs) was subsequently shown to be mediated through a novel CXCR4-iPLA2-p38 mitogen activated protein (MAP) kinase-troponin I signaling pathway [11–13]. Specifically relevant to Kelly et al. was a report that the injection of HIV gp120 resulted in a selective defect in the diastolic relaxation (lusitropic) response to adrenergic stimulation in vivo[14]. Defective adrenergic signaling is a common characteristic of both ischemic and nonischemic cardiomyopathies [16]. Previously described ‘classical’ mechanisms include decreased beta adrenergic receptor density, decreased adenylyl cyclase/protein kinase A activity, uncoupling the beta adrenergic receptor from stimulatory guanosine-5′-triphosphate (GTP)-binding protein, Gs, in conjunction with an increase in beta adrenergic receptor kinase (BARK) activity, as well as an increase in the content of the inhibitory GTP-binding protein, Gi [16]. Additional molecular insights into adrenergic signaling and diastolic function may provide an alternative mechanism for diastolic dysfunction in HIV [16]. Investigators using elegant experimental designs have provided compelling evidence for a role for p38 MAP kinase and troponin I as molecular components of signaling pathways participating in adrenergic-mediated enhancement of diastolic function. In particular, p38 MAP kinase has been reported to regulate pathways involved in the Ser 23/24 phosphorylation of troponin I [15,16]. Interestingly, a direct correlation was reported between the attenuation of the lusitropic effect of isoproterenol by the p38 MAP kinase inhibitor with blocking the phosphorylation of both p38 MAP kinase and troponin I [14]. These data are most consistent with a selective effect of HIV gp120 on p38 MAP kinase-troponin I signaling that results in attenuated adrenergic enhancement of diastolic relaxation. These clinical and experimental data are consistent with the hypothesis that HIV proteins may directly contribute to myocardial dysfunction. Kelly et al.[9] in this issue of AIDS now provide an excellent animal model with which to further explore potential molecular mechanisms contributing to myocardial dysfunction following HIV infection. Further studies into the signaling pathways stimulated by HIV proteins in the myocardium may provide novel insights and therapeutic targets relevant to HIV, as well, as other human cardiomyopathies. Acknowledgements The work was supported by NIH RO-1 #HL70565 and Department of Veterans Affairs MERIT Award. Conflicts of interest There are no conflicts of interest.

Monogenic autoimmunity.

Monogenic autoimmune syndromes provide a rare yet powerful glimpse into the fundamental mechanisms of immunologic tolerance. Such syndromes reveal not only the contribution of an individual breakpoint in tolerance but also patterns in the pathogenesis of autoimmunity. Disturbances in innate immunity, a system built for ubiquitous sensing of danger signals, tend to generate systemic autoimmunity. For example, defects in the clearance of self-antigens and chronic stimulation of type 1 interferons lead to the systemic autoimmunity seen in C1q deficiency, SPENCDI, and AGS. In contrast, disturbances of adaptive immunity, which is built for antigen specificity, tend to produce organ-specific autoimmunity. Thus, the loss of lymphocyte homeostasis, whether through defects in apoptosis, suppression, or negative selection, leads to organ-specific autoimmunity in ALPS, IPEX, and APS1. We discuss the unique mechanisms of disease in these prominent syndromes as well as how they contribute to the spectrum of organ-specific or systemic autoimmunity. The continued study of rare variants in autoimmune disease will inform future investigations and treatments directed at rare and common autoimmune diseases alike.

Foxp-Mediated Suppression of N-Cadherin Regulates Neuroepithelial Character and Progenitor Maintenance in the CNS

Neuroepithelial attachments at adherens junctions are essential for the self-renewal of neural stem and progenitor cells and the polarized organization of the developing central nervous system. The balance between stem cell maintenance and differentiation depends on the precise assembly and disassembly of these adhesive contacts, but the gene regulatory mechanisms orchestrating this process are not known. Here, we demonstrate that two Forkhead transcription factors, Foxp2 and Foxp4, are progressively expressed upon neural differentiation in the spinal cord. Elevated expression of either Foxp represses the expression of a key component of adherens junctions, N-cadherin, and promotes the detachment of differentiating neurons from the neuroepithelium. Conversely, inactivation of Foxp2 and Foxp4 function in both chick and mouse results in a spectrum of neural tube defects associated with neuroepithelial disorganization and enhanced progenitor maintenance. Together, these data reveal a Foxp-based transcriptional mechanism that regulates the integrity and cytoarchitecture of neuroepithelial progenitors.

Evaluation of subsurface defects in fiber glass composite plate using lock-in technique

In this paper, the lock-in algorithm from lock-in thermography was applied in digital shearography to evaluate the subsurface defects in fiber glass composite plate. Lock-in thermography is one of active infrared thermography which is based on propagation and reflection of thermal wave while lock-in shearography is one of optical interferometric imaging technique which is based on displacement gradient changes in a fringe pattern by mechanical or thermal loading. By applying the lock-in method to digital shearography, its sensitivity can be enhanced by phase sensitive narrow-band filtering as well as the improved signal to noise ratio, selective defects detection. Artificial defects in fiber glass composite plate were evaluated by digital shearography and lock-in shearography NDE techniques to compare their detection capacity. The experimental results confirmed that lock-in method’s efficiency on subsurface defects detection in composite plate specimen.

Bilateral Subcortical Heterotopia with Partial Callosal Agenesis in a Mouse Mutant

Cognition and behavior depend on the precise placement and interconnection of complex ensembles of neurons in cerebral cortex. Mutations that disrupt migration of immature neurons from the ventricular zone to the cortical plate have provided major insight into mechanisms of brain development and disease. We have discovered a new and highly penetrant spontaneous mutation that leads to large nodular bilateral subcortical heterotopias with partial callosal agenesis. The mutant phenotype was first detected in a colony of fully inbred BXD29 mice already known to harbor a mutation in Tlr4. Neurons confined to the heterotopias are mainly born in midgestation to late gestation and would normally have migrated into layers 2-4 of overlying neocortex. Callosal cross-sectional area and fiber number are reduced up to 50% compared with coisogenic wildtype BXD29 substrain controls. Mutants have a pronounced and highly selective defect in rapid auditory processing. The segregation pattern of the mutant phenotype is most consistent with a two-locus autosomal recessive model, and selective genotyping definitively rules out the Tlr4 mutation as a cause. The discovery of a novel mutation with strong pleiotropic anatomical and behavioral effects provides an important new resource for dissecting molecular mechanisms and functional consequences of errors of neuronal migration.

Impairment of synaptic plasticity by the stress mediator CRH involves selective destruction of thin dendritic spines via RhoA signaling

Stress is ubiquitous in modern life and exerts profound effects on cognitive and emotional functions. Thus, whereas acute stress enhances memory, longer episodes exert negative effects through as yet unresolved mechanisms. We report a novel, hippocampus-intrinsic mechanism for the selective memory defects that are provoked by stress. CRH (corticotropin-releasing hormone), a peptide released from hippocampal neurons during stress, depressed synaptic transmission, blocked activity-induced polymerization of spine actin and impaired synaptic plasticity in adult hippocampal slices. Live, multiphoton imaging demonstrated a selective vulnerability of thin dendritic spines to this stress hormone, resulting in depletion of small, potentiation-ready excitatory synapses. The underlying molecular mechanisms required activation and signaling of the actin-regulating small GTPase, RhoA. These results implicate the selective loss of dendritic spine sub-populations as a novel structural and functional foundation for the clinically important effects of stress on cognitive and emotional processes.

Macrophages from Nonobese Diabetic Mouse Have a Selective Defect in IFN-γ but Not IFN-α/β Receptor Pathway

Aberrant regulation of innate immune cells such as macrophages has been implicated in the onset and progression of type 1 diabetes (T1D). Macrophages from nonobese diabetic (NOD) mouse, an animal model of T1D, entail developmental and functional defects that are often associated with hypo-responsiveness to interferon (IFN)-γ. We aimed to uncover a mechanism underlying this phenomenon. We analyzed the receptor pathway along with the response of macrophages exposed to IFN-γ and the related IFNs such as IFN-α/β. We found that NOD macrophages failed to fully respond to IFN-γ but not to IFN-α for the production of inflammatory cytokines (e.g. TNF-α and IL-12). NOD macrophages were also resistant to apoptotic pathway induced by IFN-γ and LPS. Analyses of receptor pathway revealed that STAT1 pathway of intracellular signaling was selectively impaired in NOD macrophages exposed to IFN-γ but not to IFN-α/β. Further, these defects correlated with a low phosphorylation level of JAK2, and were related to impaired up-regulation of surface IFN-γ receptor 2 (IFN-γR2) by IFN-γ. Taken together, our results suggest that NOD macrophages have a selective defect in IFN-γ but not IFN-α/β receptor pathway. As IFN-γ and IFN-α have been implicated in the development of autoimmunity towards β-cells, such an unanticipated selectivity in IFN responsiveness may provide a new insight into the pathogenesis of T1D.