Faculty Bibliography

Lrp4, the muscle receptor for neuronal Agrin, is expressed in the hippocampus and areas involved in cognition. The function of Lrp4 in the brain, however, is unknown, as Lrp4-/- mice fail to form neuromuscular synapses and die at birth. Lrp4-/- mice, rescued for Lrp4 expression selectively in muscle, survive into adulthood and showed profound deficits in cognitive tasks that assess learning and memory. To learn whether synapses form and function aberrantly, we used electrophysiological and anatomical methods to study hippocampal CA3-CA1 synapses. In the absence of Lrp4, the organization of the hippocampus appeared normal, but the frequency of spontaneous release events and spine density on primary apical dendrites were reduced. CA3 input was unable to adequately depolarize CA1 neurons to induce long-term potentiation. Our studies demonstrate a role for Lrp4 in hippocampal function and suggest that patients with mutations in Lrp4 or auto-antibodies to Lrp4 should be evaluated for neurological deficits.

This study was aimed to determine the role and regulation of progranulin (PGRN) in the pathogenesis of inflammatory bowel diseases (IBD). Dextran sulfate sodium (DSS)-, picrylsulfonic acid (TNBS)-induced, bone marrow chimera and CD4+CD45Rb(hi) T cell transfer colitis model were established and analyzed in wild-type and several genetically-modified mice, including PGRN, IL-10 and TNFR2 deficient mice. Elevated levels of PGRN were found in colitis samples from human IBD patients and mouse colitis models in comparison to the corresponding controls. PGRN-deficient mice became highly susceptible to DSS- and TNBS-induced colitis, whereas recombinant PGRN ameliorated the pathology and reduced the histological score in both DSS and TNBS colitis models. In addition, hematopoietic-derived PGRN was critical for protection against DSS-induced colitis, and lack of PGRN signaling in CD4+ T cells also exacerbated experimental colitis. PGRN-mediated protective effect in colitis was compromised in the absence of IL-10 signaling. In addition, PGRN's effect was also largely lost in the TNFR2-deficient colitis model. Collectively, these findings not only provide the new insight into PGRN's anti-inflammatory action in vivo, but may also present PGRN and its derivatives as novel biological agent for treating IBD.

Dyneins power microtubule motility using ring-shaped, AAA-containing motor domains. Here, we report X-ray and electron microscopy (EM) structures of yeast dynein bound to different ATP analogs, which collectively provide insight into the roles of dynein's two major ATPase sites, AAA1 and AAA3, in the conformational change mechanism. ATP binding to AAA1 triggers a cascade of conformational changes that propagate to all six AAA domains and cause a large movement of the "linker," dynein's mechanical element. In contrast to the role of AAA1 in driving motility, nucleotide transitions in AAA3 gate the transmission of conformational changes between AAA1 and the linker, suggesting that AAA3 acts as a regulatory switch. Further structural and mutational studies also uncover a role for the linker in regulating the catalytic cycle of AAA1. Together, these results reveal how dynein's two major ATP-binding sites initiate and modulate conformational changes in the motor domain during motility.

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

The Golgi complex is the central organelle of the secretory pathway. It undergoes dynamic changes during the cell cycle, but how it acquires and maintains its complex structure is unclear. To address this question we have used laser nanosurgery to deplete cells of the Golgi complex and monitored its biogenesis by quantitative time-lapse microscopy and correlative electron microscopy. After Golgi depletion, ER export is inhibited and the number of ER-exit sites (ERES) is reduced and does not increase for several hours. Occasional fusion of small post-ER carriers to form the first larger structures triggers a rapid and drastic growth of Golgi precursors, due to the capacity of these structures to attract more carriers via microtubule nucleation and to stimulate ERES biogenesis. Increasing the chances of post-ER carrier fusion close to ERES by depolymerizing microtubules results in the acceleration of Golgi and ERES biogenesis. Altogether, our results propose a self-organizing principle of the early secretory pathway that integrates Golgi biogenesis, ERES biogenesis and the organization of the microtubule network by positive feedback loops.

AIMS: It is well-known that connexin43 (Cx43) forms gap junctions. We recently showed that Cx43 is also part of a protein interacting network that regulates excitability. Cardiac-specific truncation of Cx43 C-terminus (mutant "Cx43D378stop") led to lethal arrhythmias. Cx43D378stop localized to the intercalated disc (ID); cell-cell coupling was normal, but there was significant sodium current (INa) loss. We proposed that the microtubule plus-end is at the crux of the Cx43-INa relation. Yet, specific localization of relevant molecular players was prevented due to the resolution limit of fluorescence microscopy. Here, we use nanoscale imaging to establish: a) the morphology of clusters formed by the microtubule plus-end tracking protein "end binding 1" (EB1), b) their position, and that of sodium channel alpha-subunit NaV1.5, relative to N-cadherin rich sites, c) the role of Cx43 C-terminus on the above-mentioned parameters and on the location-specific function of INa. METHODS AND RESULTS: Super-resolution fluorescence localization microscopy in murine adult cardiomyocytes revealed EB1 and NaV1.5 as distinct clusters preferentially localized to N-cadherin-rich sites. Extent of co-localization decreased in Cx43D378stop cells. Macropatch and scanning patch clamp showed reduced INa exclusively at cell end, without changes in unitary conductance. Experiments in Cx43-modified HL1 cells confirmed the relation between Cx43, INa and microtubules. CONCLUSIONS: NaV1.5 and EB1 localization at cell end is Cx43-dependent. Cx43 is part of a molecular complex that determines capture of the microtubule plus-end at the ID, facilitating cargo delivery. These observations link excitability and electrical coupling through a common molecular mechanism.

IL-17 is one of the most potent and most actively investigated proinflammatory cytokines. In this study, we examined the effect of IL-17 on mesenchymal stem cells (MSCs) under the influence of inflammatory cytokines. Ironically, IL-17 dramatically enhanced the immunosuppressive effect of MSCs induced by IFNgamma and TNFalpha, revealing a novel role of IL-17 in immunosuppression. Interestingly, we found that this action of IL-17 was dependent on the promoted expression of a key immune suppressive molecule, inducible nitric oxide synthase (iNOS), in MSCs. In a concanavalin A (ConA)-induced hepatitis mouse model, we found that IL-17 also enhanced the in vivo immunosuppressive effect of MSCs in an iNOS-dependent manner. Moreover, this promoting effect of IL-17 was found to be exerted through enhancing mRNA stability by modulating the protein level of ARE/poly(U)-binding/degradation factor 1 (AUF1), a well-known factor that promotes mRNA decay. In auf1(-/-) MSCs, IFNgamma and TNFalpha could induce maximal immunosuppressive effect, both in vitro and in vivo, without the need for IL-17. Thus, our studies demonstrated that in the presence of MSCs, IL-17 promotes immunosuppression.

Chemokines are a group of small, secreted molecules that signal through G protein-coupled receptors to promote cell survival and proliferation and to provide directional guidance to migrating cells. CXCL12 is one of the most evolutionary conserved chemokines and signals through the chemokine receptor CXCR4 to guide cell migration during embryogenesis, immune cell trafficking and cancer metastasis. Here and in the accompanying poster, we provide an overview of chemokine signaling, focusing on CXCL12, and we highlight some of the different chemokine-dependent strategies used to guide migrating cells.

In this paper, we study Copy Number Variation (CNV) data. The underlying process generating CNV segments is generally assumed to be memory-less, giving rise to an exponential distribution of segment lengths. In this paper, we provide evidence from cancer patient data, which suggests that this generative model is too simplistic, and that segment lengths follow a power-law distribution instead. We conjecture a simple preferential attachment generative model that provides the basis for the observed power-law distribution. We then show how an existing statistical method for detecting cancer driver genes can be improved by incorporating the power-law distribution in the null model.