SFRP1 modulates astrocyte-to-microglia crosstalk in acute and chronic neuroinflammation.

Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.

Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis.

The vertebrate eye primordium consists of a pseudostratified neuroepithelium, the optic vesicle (OV), in which cells acquire neural retina or retinal pigment epithelium (RPE) fates. As these fates arise, the OV assumes a cup shape, influenced by mechanical forces generated within the neural retina. Whether the RPE passively adapts to retinal changes or actively contributes to OV morphogenesis remains unexplored. We generated a zebrafish Tg(E1-bhlhe40:GFP) line to track RPE morphogenesis and interrogate its participation in OV folding. We show that, in virtual absence of proliferation, RPE cells stretch and flatten, thereby matching the retinal curvature and promoting OV folding. Localized interference with the RPE cytoskeleton disrupts tissue stretching and OV folding. Thus, extreme RPE flattening and accelerated differentiation are efficient solutions adopted by fast-developing species to enable timely optic cup formation. This mechanism differs in amniotes, in which proliferation drives RPE expansion with a much-reduced need of cell flattening.

Rounded eyeballs help to optimize vision ­ but how do they acquire their distinctive shape? In animals with backbones, including humans, the eye begins to form early in development. A single layer of embryonic tissue called the optic vesicle reorganizes itself into a two-layered structure: a thin outer layer of cells, known as the retinal pigmented epithelium (RPE for short), and a thicker inner layer called the neural retina. If this process fails, the animal may be born blind or visually impaired. How this flat two-layered structure becomes round is still being investigated. In fish, studies have shown that the inner cell layer ­ the neural retina ­ generates mechanical forces that cause the developing tissue to curve inwards to form a cup-like shape. But it was unclear whether the outer layer of cells (the RPE) also contributed to this process. Moreno-Marmol et al. were able to investigate this question by genetically modifying zebrafish to make all new RPE cells fluoresce. Following the early development of the zebrafish eye under a microscope revealed that RPE cells flattened themselves into long thin structures that stretched to cover the entire neural retina. This change was made possible by the cell's internal skeleton reorganizing. In fact, preventing this reorganization stopped the RPE cells from flattening, and precluded the optic cup from acquiring its curved shape. The results thus confirmed a direct role for the RPE in generating curvature. The entire process did not require the RPE to produce new cells, allowing the curved shape to emerge in just a few hours. This is a major advantage for fast-developing species such as zebrafish. In species whose embryos develop more slowly, such as mice and humans, the RPE instead grows by producing additional cells ­ a process that takes many days. The development of the eye thus shows how various species use different evolutionary approaches to achieve a common goal.

Sfrp1 deficiency makes retinal photoreceptors prone to degeneration.

Millions of individuals worldwide suffer from impaired vision, a condition with multiple origins that often impinge upon the light sensing cells of the retina, the photoreceptors, affecting their integrity. The molecular components contributing to this integrity are however not yet fully understood. Here we have asked whether Secreted Frizzled Related Protein 1 (SFRP1) may be one of such factors. SFRP1 has a context-dependent function as modulator of Wnt signalling or of the proteolytic activity of A Disintegrin And Metalloproteases (ADAM) 10, a main regulator of neural cell-cell communication. We report that in Sfrp1-/- mice, the outer limiting membrane (OLM) is discontinuous and the photoreceptors disorganized and more prone to light-induced damage. Sfrp1 loss significantly enhances the effect of the Rpe65Leu450Leu genetic variant -present in the mouse genetic background- which confers sensitivity to light-induced stress. These alterations worsen with age, affect visual function and are associated to an increased proteolysis of Protocadherin 21 (PCDH21), localized at the photoreceptor outer segment, and N-cadherin, an OLM component. We thus propose that SFRP1 contributes to photoreceptor fitness with a mechanism that involves the maintenance of OLM integrity. These conclusions are discussed in view of the broader implication of SFRP1 in neurodegeneration and aging.

Elevated levels of Secreted-Frizzled-Related-Protein 1 contribute to Alzheimer's disease pathogenesis.

The deposition of aggregated amyloid-ß peptides derived from the pro-amyloidogenic processing of the amyloid precurson protein (APP) into characteristic amyloid plaques (APs) is distinctive to Alzheimer's disease (AD). Alternative APP processing via the metalloprotease ADAM10 prevents amyloid-ß formation. We tested whether downregulation of ADAM10 activity by its secreted endogenous inhibitor secreted-frizzled-related protein 1 (SFRP1) is a common trait of sporadic AD. We demonstrate that SFRP1 is significantly increased in the brain and cerebrospinal fluid of patients with AD, accumulates in APs and binds to amyloid-ß, hindering amyloid-ß protofibril formation. Sfrp1 overexpression in an AD-like mouse model anticipates the appearance of APs and dystrophic neurites, whereas its genetic inactivation or the infusion of α-SFRP1-neutralizing antibodies favors non-amyloidogenic APP processing. Decreased Sfrp1 function lowers AP accumulation, improves AD-related histopathological traits and prevents long-term potentiation loss and cognitive deficits. Our study unveils SFRP1 as a crucial player in AD pathogenesis and a promising AD therapeutic target.

Antigen phagocytosis by B cells is required for a potent humoral response.

Successful vaccines rely on activating a functional humoral response that results from promoting a proper germinal center (GC) reaction. Key in this process is the activation of follicular B cells that need to acquire antigens and to present them to cognate CD4 T cells. Here, we report that follicular B cells can phagocytose large antigen-coated particles, a process thought to be exclusive of specialized antigen-presenting cells such as macrophages and dendritic cells. We show that antigen phagocytosis by B cells is BCR-driven and mechanistically dependent on the GTPase RhoG. Using Rhog-/- mice, we show that phagocytosis of antigen by B cells is important for the development of a strong GC response and the generation of high-affinity class-switched antibodies. Importantly, we show that the potentiation effect of alum, a common vaccine adjuvant, requires direct phagocytosis of alum-antigen complexes by B cells. These data suggest a new avenue for vaccination approaches by aiming to deliver 1-3 µm size antigen particles to follicular B cells.

Meis1 coordinates a network of genes implicated in eye development and microphthalmia.

Microphthalmos is a rare congenital anomaly characterized by reduced eye size and visual deficits of variable degree. Sporadic and hereditary microphthalmos have been associated with heterozygous mutations in genes fundamental for eye development. Yet, many cases are idiopathic or await the identification of molecular causes. Here we show that haploinsufficiency of Meis1, which encodes a transcription factor with evolutionarily conserved expression in the embryonic trunk, brain and sensory organs, including the eye, causes microphthalmic traits and visual impairment in adult mice. By combining analysis of Meis1 loss-of-function and conditional Meis1 functional rescue with ChIP-seq and RNA-seq approaches we show that, in contrast to its preferential association with Hox-Pbx BSs in the trunk, Meis1 binds to Hox/Pbx-independent sites during optic cup development. In the eye primordium, Meis1 coordinates, in a dose-dependent manner, retinal proliferation and differentiation by regulating genes responsible for human microphthalmia and components of the Notch signaling pathway. In addition, Meis1 is required for eye patterning by controlling a set of eye territory-specific transcription factors, so that in Meis1(-/-) embryos boundaries among the different eye territories are shifted or blurred. We propose that Meis1 is at the core of a genetic network implicated in eye patterning/microphthalmia, and represents an additional candidate for syndromic cases of these ocular malformations.

Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons.

Although human olfactory mucosa derived cells (OMC) have been used in animal models and clinical trials with CNS repair purposes, the exact identity of these cells in culture with respect to their tissue of origin is not fully understood and their neuroregenerative capacity in vitro has not yet been demonstrated. In this study we have compared human OMC with human ensheathing glia from olfactory bulb (OB) and human fibroblasts from skin and lung. Our results indicate that these different cultured cell types exhibit considerable overlap of antigenic markers such that it is presently not possible to distinguish them immunocytochemically. However, in rat retinal ganglion neuron coculture assays the axonal regenerative activity of OMC and OB ensheathing glia was dramatically higher than that exhibited by all fibroblast samples, confirming neuroregenerative activity as a unique property shared by cultured cells derived from the human olfactory system.

Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration.

Olfactory ensheathing glia (OEG) cells are known to facilitate repair following axotomy of adult neurons, although the molecular mechanisms involved are not fully understood. We previously identified plasminogen activator inhibitor-1 (PAI-1), proteinase-activated receptor-1 (PAR-1), and thrombomodulin (TM) as candidates to regulate rat OEG-dependent axonal regeneration. In this study, we have validated the involvement of these proteins in promoting axonal regeneration by immortalized human OEGs. We studied the effect of silencing these proteins in OEGs on their capacity to promote the regeneration of severed adult retinal ganglion cells (RGCs) axons. Our results support the role of glial PAI-1 as a downstream effector of PAR-1 in promoting axon regeneration. In contrast, we found that TM inhibits OEG induced-axonal regeneration. We also assessed the signaling pathways downstream of PAR-1 that might modulate PAI-1 expression, observing that specifically inhibiting Gα(i), Rho kinase, or PLC and PKC downregulated the expression of PAI-1 in OEGs, with a concomitant reduction in OEG-dependent axon regeneration in adult RGCs. Our findings support an important role for the thrombin system in regulating adult axonal regeneration by OEGs.

A neuroregenerative human ensheathing glia cell line with conditional rapid growth.

Ensheathing glia have been demonstrated to have neuroregenerative properties but this cell type from human sources has not been extensively studied because tissue samples are not easily obtained, primary cultures are slow growing, and human cell lines are not available. We previously isolated immortalized ensheathing glia by gene transfer of BMI1 and telomerase catalytic subunit into primary cultures derived from olfactory bulbs of an elderly human cadaver donor. These cells escape the replicative senescence characteristic of primary human cells while conserving antigenic and neuroregenerative properties of ensheathing glia, but their low proliferative rate in culture complicates their utility as cell models and their application for preclinical cell therapy experiments. In this study we describe the use of a conditional SV40 T antigen (TAg) transgene to generate human ensheathing glia cell lines, which are easy to maintain due to their robust growth in culture. Although these fast growing clones exhibited polyploid karyotypes frequently observed in cells immortalized by TAg, they did not acquire a transformed phenotype, all of them maintaining neuroregenerative capacity and antigenic markers typical of ensheathing glia. These markers were also retained even after elimination of the TAg transgene using Cre/LoxP technology, although the cells died shortly after, confirming that their survival depended on the presence of the immortalizing genes. We have also demonstrated here the feasibility of using these human cell lines in animal models by genetically marking the cells with GFP and implanting them into the injured spinal cord of immunosuppressed rats. Our conditionally immortalized human ensheathing glia cell lines will thus serve as useful tools for advancing cell therapy approaches and understanding neuroregenerative mechanisms of this unique cell type.