Roland E. Kälin

An unconventional role for miRNA: let-7 activates Toll-like receptor 7 and causes neurodegeneration

Activation of innate immune receptors by host-derived factors exacerbates CNS damage, but the identity of these factors remains elusive. We uncovered an unconventional role for the microRNA let-7, a highly abundant regulator of gene expression in the CNS, in which extracellular let-7 activates the RNA-sensing Toll-like receptor (TLR) 7 and induces neurodegeneration through neuronal TLR7. Cerebrospinal fluid (CSF) from individuals with Alzheimers disease contains increased amounts of let-7b, and extracellular introduction of let-7b into the CSF of wild-type mice by intrathecal injection resulted in neurodegeneration. Mice lacking TLR7 were resistant to this neurodegenerative effect, but this susceptibility to let-7 was restored in neurons transfected with TLR7 by intrauterine electroporation of Tlr7−/− fetuses. Our results suggest that microRNAs can function as signaling molecules and identify TLR7 as an essential element in a pathway that contributes to the spread of CNS damage.

Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline

The pathology of Alzheimers disease has an inflammatory component that is characterized by upregulation of proinflammatory cytokines, particularly in response to amyloid-β (Aβ). Using the APPPS1 Alzheimers disease mouse model, we found increased production of the common interleukin-12 (IL-12) and IL-23 subunit p40 by microglia. Genetic ablation of the IL-12/IL-23 signaling molecules p40, p35 or p19, in which deficiency of p40 or its receptor complex had the strongest effect, resulted in decreased cerebral amyloid load. Although deletion of IL-12/IL-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in a reduction of cerebral amyloid load in APPPS1 mice. Furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced the concentration of soluble Aβ species and reversed cognitive deficits in aged APPPS1 mice. The concentration of p40 was also increased in the cerebrospinal fluid of subjects with Alzheimers disease, which suggests that inhibition of the IL-12/IL-23 pathway may attenuate Alzheimers disease pathology and cognitive deficits.

A genetic Xenopus laevis tadpole model to study lymphangiogenesis

Lymph vessels control fluid homeostasis, immunity and metastasis. Unraveling the molecular basis of lymphangiogenesis has been hampered by the lack of a small animal model that can be genetically manipulated. Here, we show that Xenopus tadpoles develop lymph vessels from lymphangioblasts or, through transdifferentiation, from venous endothelial cells. Lymphangiography showed that these lymph vessels drain lymph, through the lymph heart, to the venous circulation. Morpholino-mediated knockdown of the lymphangiogenic factor Prox1 caused lymph vessel defects and lymphedema by impairing lymphatic commitment. Knockdown of vascular endothelial growth factor C (VEGF-C) also induced lymph vessel defects and lymphedema, but primarily by affecting migration of lymphatic endothelial cells. Knockdown of VEGF-C also resulted in aberrant blood vessel formation in tadpoles. This tadpole model offers opportunities for the discovery of new regulators of lymphangiogenesis.

miR-31 Functions as a Negative Regulator of Lymphatic Vascular Lineage-Specific Differentiation In Vitro and Vascular Development In Vivo

ABSTRACT The lymphatic vascular system maintains tissue fluid homeostasis, helps mediate afferent immune responses, and promotes cancer metastasis. To address the role microRNAs (miRNAs) play in the development and function of the lymphatic vascular system, we defined the in vitro miRNA expression profiles of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) and identified four BVEC signature and two LEC signature miRNAs. Their vascular lineage-specific expression patterns were confirmed in vivo by quantitative real-time PCR and in situ hybridization. Functional characterization of the BVEC signature miRNA miR-31 identified a novel BVEC-specific posttranscriptional regulatory mechanism that inhibits the expression of lymphatic lineage-specific transcripts in vitro. We demonstrate that suppression of lymphatic differentiation is partially mediated via direct repression of PROX1, a transcription factor that functions as a master regulator of lymphatic lineage-specific differentiation. Finally, in vivo studies of Xenopus and zebrafish demonstrated that gain of miR-31 function impaired venous sprouting and lymphatic vascular development, thus highlighting the importance of miR-31 as a negative regulator of lymphatic development. Collectively, our findings identify miR-31 is a potent regulator of vascular lineage-specific differentiation and development in vertebrates.

An in vivo chemical library screen in Xenopus tadpoles reveals novel pathways involved in angiogenesis and lymphangiogenesis

Angiogenesis and lymphangiogenesis are essential for organogenesis but also play important roles in tissue regeneration, chronic inflammation, and tumor progression. Here we applied in vivo forward chemical genetics to identify novel compounds and biologic mechanisms involved in (lymph)angiogenesis in Xenopus tadpoles. A novel 2-step screening strategy involving a simple phenotypic read-out (edema formation or larval lethality) followed by semiautomated in situ hybridization was devised and used to screen an annotated chemical library of 1280 bioactive compounds. We identified 32 active compounds interfering with blood vascular and/or lymphatic development in Xenopus. Selected compounds were also tested for activities in a variety of endothelial in vitro assays. Finally, in a proof-of-principle study, the adenosine A1 receptor antagonist 7-chloro-4-hydroxy-2-phenyl-1,8-naphthyridine, an inhibitor of blood vascular and lymphatic development in Xenopus, was shown to act also as a potent antagonist of VEGFA-induced adult neovascularization in mice. Taken together, the present chemical library screening strategy in Xenopus tadpoles represents a rapid and highly efficient approach to identify novel pathways involved in (lymph)angiogenesis. In addition, the recovered compounds represent a rich resource for in-depth analysis, and their drug-like features will facilitate further evaluation in preclinical models of inflammation and cancer metastasis.

The FGFRL1 receptor is shed from cell membranes, binds fibroblast growth factors (FGFs), and antagonizes FGF signaling in Xenopus embryos.

FGFRL1 (fibroblast growth factor receptor like 1) is the fifth and most recently discovered member of the fibroblast growth factor receptor (FGFR) family. With up to 50% amino acid similarity, its extracellular domain closely resembles that of the four conventional FGFRs. Its intracellular domain, however, lacks the split tyrosine kinase domain needed for FGF-mediated signal transduction. During embryogenesis of the mouse, FGFRL1 is essential for the development of parts of the skeleton, the diaphragm muscle, the heart, and the metanephric kidney. Since its discovery, it has been hypothesized that FGFRL1 might act as a decoy receptor for FGF ligands. Here we present several lines of evidence that support this notion. We demonstrate that the FGFRL1 ectodomain is shed from the cell membrane of differentiating C2C12 myoblasts and from HEK293 cells by an as yet unidentified protease, which cuts the receptor in the membrane-proximal region. As determined by ligand dot blot analysis, cell-based binding assays, and surface plasmon resonance analysis, the soluble FGFRL1 ectodomain as well as the membrane-bound receptor are capable of binding to some FGF ligands with high affinity, including FGF2, FGF3, FGF4, FGF8, FGF10, and FGF22. We furthermore show that ectopic expression of FGFRL1 in Xenopus embryos antagonizes FGFR signaling during early development. Taken together, our data provide strong evidence that FGFRL1 is indeed a decoy receptor for FGFs.

CNS myeloid cells critically regulate heat hyperalgesia

&NA; Activation of non‐neuronal microglia is thought to play a causal role in spinal processing of neuropathic pain. To specifically investigate microglia‐mediated effects in a model of neuropathic pain and overcome the methodological limitations of previous approaches exploring microglia function upon nerve injury, we selectively ablated resident microglia by intracerebroventricular ganciclovir infusion into male CD11b‐HSVTK‐transgenic mice, which was followed by a rapid, complete, and persistent (23 weeks) repopulation of the CNS by peripheral myeloid cells. In repopulated mice that underwent sciatic nerve injury, we observed a normal response to mechanical stimuli, but an absence of thermal hypersensitivity ipsilateral to the injured nerve. Furthermore, we found that neuronal expression of calcitonin gene‐related peptide (CGRP), which is a marker of neurons essential for heat responses, was diminished in the dorsal horn of the spinal cord in repopulated mice. These findings identify distinct mechanisms for heat and mechanical hypersensitivity and highlight a crucial contribution of CNS myeloid cells in the facilitation of noxious heat.