Engulfed by Glia: Glial Pruning in Development, Function, and Injury across Species.

Phagocytic activity of glial cells is essential for proper nervous system sculpting, maintenance of circuitry, and long-term brain health. Glial engulfment of apoptotic cells and superfluous connections ensures that neuronal connections are appropriately refined, while clearance of damaged projections and neurotoxic proteins in the mature brain protects against inflammatory insults. Comparative work across species and cell types in recent years highlights the striking conservation of pathways that govern glial engulfment. Many signaling cascades used during developmental pruning are re-employed in the mature brain to "fine tune" synaptic architecture and even clear neuronal debris following traumatic events. Moreover, the neuron-glia signaling events required to trigger and perform phagocytic responses are impressively conserved between invertebrates and vertebrates. This review offers a compare-and-contrast portrayal of recent findings that underscore the value of investigating glial engulfment mechanisms in a wide range of species and contexts.

Mutations in pre-mRNA processing factors 3, 8, and 31 cause dysfunction of the retinal pigment epithelium.

Mutations in the ubiquitously expressed pre-mRNA processing factors 3, 8, and 31 (PRPF3, PRPF8, and PRPF31) cause nonsyndromic dominant retinitis pigmentosa in humans, an inherited retinal degeneration. It is unclear what mechanisms, or which cell types of the retina, are affected. Transgenic mice with the human mutations in these genes display late-onset morphological changes in the retinal pigment epithelium (RPE). To determine whether the observed morphological changes are preceded by abnormal RPE function, we investigated its phagocytic function in Prpf3(T494M/T494M), Prpf8(H2309P/H2309P), and Prpf31(+/-) mice. We observe decreased phagocytosis in primary RPE cultures from mutant mice, and this is replicated by shRNA-mediated knockdown of PRPF31 in human ARPE-19 cells. The diurnal rhythmicity of phagocytosis is almost lost, indicated by the marked attenuation of the phagocytic burst 2 hours after light onset. The strength of adhesion between RPE apical microvilli and photoreceptor outer segments also declined during peak adhesion in all mutants. In all models, at least one of the receptors involved in binding and internalization of shed photoreceptor outer segments was subjected to changes in localization. Although the mechanism underlying these changes in RPE function is yet to be elucidated, these data are consistent with the mouse RPE being the primary cell affected by mutations in the RNA splicing factors, and these changes occur at an early age.

Galectin-3 Promotes Müller Glia Clearance Phagocytosis via MERTK and Reduces Harmful Müller Glia Activation in Inherited and Induced Retinal Degeneration.

Clearance phagocytosis is a documented function of Müller glia in the retina. However, the molecular mechanisms of Müller glia phagocytosis remain largely undefined. Here, we show that extracellular galectin-3 and protein S promote clearance phagocytosis by immortalized human MIO-M1 Müller cells in an additive, saturable manner. Galectin-3 promotes phagocytosis by primary Müller glia from wild-type (WT) mice but not from mice that lack the engulfment receptor MERTK and therefore develop postnatal photoreceptor degeneration. Probing a possible functional link between Müller galectin-3 and MERTK, we discovered that mertk -/- Müller glia in situ show excess galectin-3 at postnatal day 20 (P20), an age prior to detectable photoreceptor degeneration. Moreover, double knockout (DKO) mice lacking both galectin-3 and MERTK show increased activation of Müller cells (but not of microglia) at P20 and more pronounced photoreceptor loss at P35 compared to mice lacking MERTK alone. Exploring the well-established sodium iodate injury model, we also found more severe activation specifically of Müller glia, and worse retinal damage in mice lacking galectin-3 compared to WT mice. Indeed, galectin-3 deficiency significantly increased sensitivity to injury, yielding Müller activation and retinal damage at a sodium iodate concentration that had no effect on the WT retina. Altogether, our results from both inherited and acutely induced models of retinal degeneration agree that eliminating galectin-3 exacerbates Müller cell activation and retinal degeneration. These data identify an important protective role for the MERTK ligand galectin-3 in the retina in restraining Müller glia activation.

Microglia Inhibition Delays Retinal Degeneration Due to MerTK Phagocytosis Receptor Deficiency.

Retinitis Pigmentosa (RP) is a group of inherited retinal diseases characterized by progressive loss of rod followed by cone photoreceptors. An especially early onset form of RP with blindness in teenage years is caused by mutations in mertk, the gene encoding the clearance phagocytosis receptor Mer tyrosine kinase (MerTK). The cause for blindness in mutant MerTK-associated RP (mutMerTK-RP) is the failure of retinal pigment epithelial cells in diurnal phagocytosis of spent photoreceptor outer segment debris. However, the early onset and very fast progression of degeneration in mutMerTK-RP remains unexplained. Here, we explored the role of microglia in the Royal College of Surgeons (RCS) rat model of mutMerTK-RP. We found elevated levels of inflammatory cytokines and CD68 microglia activation marker, and more ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia in RCS retina when compared to wild-type retina as early as postnatal day 14 (P14). Strikingly, renewal of photoreceptor outer segments in P14 wild-type rat retina is still immature with low levels of RPE phagocytosis implying that at this early age lack of this process in RCS rats is unlikely to distress photoreceptors. Although the total number of Iba-1 positive retinal microglia remains constant from P14 to P30, we observed increasing numbers of microglia in the outer retina from P20 implying migration to the outer retina before onset of photoreceptor cell death at ~P25. Iba-1 and CD68 levels also increase in the retina during this time period suggesting microglia activation. To determine whether microglia affect the degenerative process, we suppressed retinal microglia in vivo using tamoxifen or a combination of tamoxifen and liposomal clodronate. Treatments partly prevented elevation of Iba-1 and CD68 and relocalization of microglia. Moreover, treatments led to partial but significant retention of photoreceptor viability and photoreceptor function. We conclude that loss of the phagocytosis receptor MerTK causes microglia activation and relocalization in the retina before lack of RPE phagocytosis causes overt retinal degeneration, and that microglia activities accelerate loss of photoreceptors in mutMerTK-RP. These results suggest that therapies targeting microglia may delay onset and slow the progression of this blinding disease.

Non-invasive in vivo fluorescence imaging of apoptotic retinal photoreceptors.

Phosphatidylserine externalization is an early molecular signature for apoptosis. In many retinal degenerative diseases, photoreceptor neurons die by apoptosis. Here, we report utility of the phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine (Zn-DPA) with Texas-red (PSVue-550) in transiently labeling apoptotic photoreceptors in living pigmented or albino rats and mice with retinal degeneration. Applying PSVue-550 as eyedrop is non-toxic and eliminates need for intraocular injection. PSVue-550 fluorescence specifically and transiently labeling dying retinal photoreceptors is detectable in anesthetized animals using standard retinal or whole small animal imaging systems. Importantly, prior PSVue-550 eyedrop administration and imaging does not affect repeat testing. Altogether, our results establish PSVue-550 imaging as a completely non-invasive method that provides the opportunity to longitudinally monitor retinal photoreceptor cell death in preclinical studies.

Leukocyte-specific protein 1 links TNF receptor-associated factor 1 to survival signaling downstream of 4-1BB in T cells.

4-1BB is a member of the TNFR superfamily, which contributes to the activation of signaling pathways required for the survival of activated and memory T cells. We have shown previously that TRAF1, an adaptor protein recruited to 4-1BB, is required for 4-1BB-mediated CD8 T cell survival in vivo. With the use of a proteomics approach in primary T cells, we have identified LSP1 as a novel protein recruited to the 4-1BB signaling complex in a TRAF1-dependent manner. Further characterization of the interaction between TRAF1 and LSP1 revealed that LSP1 requires the TRAF-N domain of TRAF1 for direct association. Similarly to TRAF1(-/-) T cells, LSP1(-/-) T cells exhibit impaired ERK activation following stimulation through 4-1BB and consequently, are unable to down-modulate expression of the proapoptotic Bcl-2 family member Bim. Moreover, we demonstrate that the absence of LSP1 expression leads to defective expansion and survival of T cells in response to 4-1BB stimulation. Thus, we have identified LSP1 as a new mediator involved in 4-1BB signaling and T cell survival. Collectively, our work shows that TRAF1 and LSP1 cooperate downstream of 4-1BB to activate ERK signaling and down-modulate the levels of Bim leading to enhanced T cell survival.

Wnt4 enhances murine hematopoietic progenitor cell expansion through a planar cell polarity-like pathway.

BACKGROUND: While the role of canonical (ß-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while ß-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. CONCLUSIONS/SIGNIFICANCE: Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis through a pathway involving Wnt4, Fz6, Rac1 and Jnk kinases.