Chemokine-like Orion is involved in the transformation of glial cells into phagocytes in different developmental neuronal remodeling paradigms.

During animal development, neurons often form exuberant or inappropriate axons and dendrites at early stages, followed by the refinement of neuronal circuits at late stages. Neural circuit refinement leads to the production of neuronal debris in the form of neuronal cell corpses, fragmented axons and dendrites, and pruned synapses requiring disposal. Glial cells act as predominant phagocytes during neuronal remodeling and degeneration, and crucial signaling pathways between neurons and glia are necessary for the execution of phagocytosis. Chemokine-like mushroom body neuron-secreted Orion is essential for astrocyte infiltration into the γ axon bundle leading to γ axon pruning. Here, we show a role of Orion in debris engulfment and phagocytosis in Drosophila. Interestingly, Orion is involved in the overall transformation of astrocytes into phagocytes. In addition, analysis of several neuronal paradigms demonstrates the role of Orion in eliminating both peptidergic vCrz+ and PDF-Tri neurons via additional phagocytic glial cells like cortex and/or ensheathing glia. Our results suggest that Orion is essential for phagocytic activation of astrocytes, cortex and ensheathing glia, and point to Orion as a trigger of glial infiltration, engulfment and phagocytosis.

The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons.

Phagocytic clearance of degenerating neurons is triggered by "eat-me" signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, we show that the Drosophila chemokine-like protein Orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of Orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the Orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between Orion and human immunomodulatory proteins are important for Orion function. Thus, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.

Neuron-glia crosstalk in neuronal remodeling and degeneration: Neuronal signals inducing glial cell phagocytic transformation in Drosophila.

Neuronal remodeling is a conserved mechanism that eliminates unwanted neurites and can include the loss of cell bodies. In these processes, a key role for glial cells in events from synaptic pruning to neuron elimination has been clearly identified in the last decades. Signals sent from dying neurons or neurites to be removed are received by appropriate glial cells. After receiving these signals, glial cells infiltrate degenerating sites and then, engulf and clear neuronal debris through phagocytic mechanisms. There are few identified or proposed signals and receptors involved in neuron-glia crosstalk, which induces the transformation of glial cells to phagocytes during neuronal remodeling in Drosophila. Many of these signaling pathways are conserved in mammals. Here, we particularly emphasize the role of Orion, a recently identified neuronal CX3 C chemokine-like secreted protein, which induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling. Although, chemokine signaling was not described previously in insects we propose that chemokine-like involvement in neuron/glial cell interaction is an evolutionarily ancient mechanism.

Htt is a repressor of Abl activity required for APP-induced axonal growth.

Huntington's disease is a progressive autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract at the N-terminus of a large cytoplasmic protein. The Drosophila huntingtin (htt) gene is widely expressed during all developmental stages from embryos to adults. However, Drosophila htt mutant individuals are viable with no obvious developmental defects. We asked if such defects could be detected in htt mutants in a background that had been genetically sensitized to reveal cryptic developmental functions. Amyloid precursor protein (APP) is linked to Alzheimer's disease. Appl is the Drosophila APP ortholog and Appl signaling modulates axon outgrowth in the mushroom bodies (MBs), the learning and memory center in the fly, in part by recruiting Abl tyrosine kinase. Here, we find that htt mutations suppress axon outgrowth defects of αß neurons in Appl mutant MB by derepressing the activity of Abl. We show that Abl is required in MB αß neurons for their axon outgrowth. Importantly, both Abl overexpression and lack of expression produce similar phenotypes in the MBs, indicating the necessity of tightly regulating Abl activity. We find that Htt behaves genetically as a repressor of Abl activity, and consistent with this, in vivo FRET-based measurements reveal a significant increase in Abl kinase activity in the MBs when Htt levels are reduced. Thus, Appl and Htt have essential but opposing roles in MB development, promoting and suppressing Abl kinase activity, respectively, to maintain the appropriate intermediate level necessary for axon growth.

Nuclear receptors and Drosophila neuronal remodeling.

During the development of both vertebrates and invertebrates, neurons undergo a crucial remodeling process that is necessary for their new function. Neuronal remodeling is composed of two stages: first, axons and dendrites are pruned without the loss of the cell body; later, this process is most commonly followed by a regrowth step. Holometabolous insects like the fruitfly Drosophila exhibit striking differences between their larval and adult stages. These neuronal remodeling processes occur during metamorphosis, the period of transformation from a larva to an adult. All axon and dendrite pruning events ultimately depend on the EcR nuclear receptor. Its ligand, the steroid molting hormone ecdysone, binds to heteromeric receptors comprising the nuclear receptor ECR and USP, and this complex regulates target genes involved in neuronal remodeling. Here we review the nuclear receptor-mediated genetic control of the main neuronal remodeling events described so far in Drosophila. These events consist of neurite degeneration in the mushroom bodies (MBs: the brain memory center) and in the dendritic arborizing sensory neurons, of neurite retraction or small scale elimination in the thoracic ventral neurosecretory cells, in the olfactory circuits and in the neuromuscular junction. MB axon regrowth after pruning and the role of MB neuron remodeling in memory formation are also reviewed. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Mushroom body neuronal remodelling is necessary for short-term but not for long-term courtship memory in Drosophila.

The remodelling of neurons during their development is considered necessary for their normal function. One fundamental mechanism involved in this remodelling process in both vertebrates and invertebrates is axon pruning. A well-documented case of such neuronal remodelling is the developmental axon pruning of mushroom body γ neurons that occurs during metamorphosis in Drosophila. The γ neurons undergo pruning of larval-specific dendrites and axons at metamorphosis, followed by their regrowth as adult-specific dendrites and axons. We recently revealed a molecular cascade required for this pruning. The nuclear receptor ftz-f1 activates the expression of the steroid hormone receptor EcR-B1, a key component for γ remodelling, and represses expression of Hr39, an ftz-f1 homologous gene. If ectopically expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. The mushroom bodies are a bilaterally symmetric structure in the larval and adult brain and are involved in the processing of different types of olfactory memory. How memory is affected in pruning-deficient adult flies that possess larval-stage neuronal circuitry will help to explain the functional role of neuron remodelling. Flies overexpressing Hr39 are viable as adults and make it possible to assess the requirement for wild-type mushroom body pruning in memory. While blocking mushroom body neuron remodelling impaired memory after short-term courtship conditioning, long-term memory was normal. These results show that larval pruning is necessary for adult memory and that expression of courtship short-term memory and long-term memory may be parallel and independent.

Axonal chemokine-like Orion induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling.

The remodeling of neurons is a conserved fundamental mechanism underlying nervous system maturation and function. Astrocytes can clear neuronal debris and they have an active role in neuronal remodeling. Developmental axon pruning of Drosophila memory center neurons occurs via a degenerative process mediated by infiltrating astrocytes. However, how astrocytes are recruited to the axons during brain development is unclear. Using an unbiased screen, we identify the gene requirement of orion, encoding for a chemokine-like protein, in the developing mushroom bodies. Functional analysis shows that Orion is necessary for both axonal pruning and removal of axonal debris. Orion performs its functions extracellularly and bears some features common to chemokines, a family of chemoattractant cytokines. We propose that Orion is a neuronal signal that elicits astrocyte infiltration and astrocyte-driven axonal engulfment required during neuronal remodeling in the Drosophila developing brain.

Downregulation of otospiralin, a novel inner ear protein, causes hair cell degeneration and deafness.

Mesenchymal nonsensory regions of the inner ear are important structures surrounding the neurosensory epithelium that are believed to participate in the ionic homeostasis of the cochlea and vestibule. We report here the discovery of otospiralin, an inner ear-specific protein that is produced by fibrocytes from these regions, including the spiral ligament and spiral limbus in the cochlea and the maculae and semicircular canals in the vestibule. Otospiralin is a novel 6.4 kDa protein of unknown function that shares a protein motif with the gag p30 core shell nucleocapsid protein of type C retroviruses. To evaluate its functional importance, we downregulated otospiralin by cochlear perfusion of antisense oligonucleotides in guinea pigs. This led to a rapid threshold elevation of the compound action potentials and irreversible deafness. Cochlear examination by transmission electron microscopy revealed hair cell loss and degeneration of the organ of Corti. This demonstrates that otospiralin is essential for the survival of the neurosensory epithelium.

Identification of beta-carotene 15, 15'-monooxygenase as a peroxisome proliferator-activated receptor target gene.

Beta-carotene 15,15'-monooxygenase (BCM) catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids. We wished to determine the factors underlying the transcriptional regulation of this gene. After cloning of the 40 kilobase pair (kbp) mouse Bcm gene and determination of its genomic organization, analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites including TATA box, a peroxisome proliferator response element (PPRE), AP2, and bHLH. The 2 kb fragment drove specific luciferase gene expression in vitro only in cell lines that express BCM (TC7, PF11, and monkey retinal pigment epithelium). Nucleotides -41 to +163, and -60 to +163 drove basal and specific Bcm transcriptional activity, respectively. Site-directed mutagenesis and gel shift experiments demonstrate that PPRE was essential for Bcm promoter specificity and that the peroxisome proliferator activated receptor (PPAR) gamma (PPARgamma) specifically binds to this element. Furthermore, cotransfection experiments and pharmacological treatments in vitro, using the specific PPARgamma agonists LY17883 and ciglitazone, demonstrate that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor-alpha heterodimer. Treatment of mice with the PPARalpha/gamma agonist WY14643 increases BCM protein expression in liver. Thus PPAR is a key transcription factor for the transcriptional regulation of the Bcm gene, suggesting a broader function for PPARs in the regulation of carotenoid metabolism metabolism that is consistent with their established role in neutral lipid metabolism and transport.

Guidance of Drosophila Mushroom Body Axons Depends upon DRL-Wnt Receptor Cleavage in the Brain Dorsomedial Lineage Precursors.

In vivo axon pathfinding mechanisms in the neuron-dense brain remain relatively poorly characterized. We study the Drosophila mushroom body (MB) axons, whose α and ß branches connect to different brain areas. We show that the Ryk family WNT5 receptor, DRL (derailed), which is expressed in the dorsomedial lineages, brain structure precursors adjacent to the MBs, is required for MB α branch axon guidance. DRL acts to capture and present WNT5 to MB axons rather than transduce a WNT5 signal. DRL's ectodomain must be cleaved and shed to guide α axons. DRL-2, another Ryk, is expressed within MB axons and functions as a repulsive WNT5 signaling receptor. Finally, our biochemical data support the existence of a ternary complex composed of the cleaved DRL ectodomain, WNT5, and DRL-2. Thus, the interaction of MB-extrinsic and -intrinsic Ryks via their common ligand acts to guide MB α axons.

Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog.

PURPOSE: To assess the efficacy of AAV-mediated gene therapy to restore vision in a large number of RPE65(-/-) dogs and to determine whether systemic and local side effects are caused by the treatment. METHODS: Normal RPE65 dog cDNA was subcloned into an rAAV vector under control of a cytomegalovirus promoter, and an AAV.GFP control vector was also produced with the titers 2 x 10(12) particles/mL and 2 x 10(10) transducing U/mL, respectively. RPE65(-/-) dogs, aged 4 to 30 months were treated with subretinal injections of the AAV.RPE65 and control vectors, respectively, in each eye, and three 24- to 30-month-old normal control dogs with the latter. Baseline and postoperative systemic and ophthalmic examinations, blood screenings, vision testing, and electroretinography (ERG) were performed. Two RPE65(-/-) dogs were killed at 3 and 6 months after treatment for morphologic examination of the retinas. RESULTS: RPE65(-/-) dogs were practically blind from birth with nonrecordable or low-amplitude ERGs. Construct injections or sham surgeries were performed in 28 eyes; 11 were injected subretinally with the AAV.RPE65 construct. ERGs at 3 months after surgery showed that in the latter eyes, dark-adapted b-wave amplitudes recovered to an average of 28% of normal, and light adapted b-wave amplitudes to 32% of normal. ERG amplitudes were not reduced during a 6- to 9-month follow-up. No systemic side effects were observed, but uveitis developed in nine AAV.RPE65-treated eyes. No uveitis was observed in the eyes treated with the control vector. Immunocytochemistry showed expression of RPE65 in the retinal pigment epithelium (RPE) of AAV.RPE65-treated eyes. Fluorescence microscopy showed expression of green fluorescent protein (GFP) in the RPE and, to a lesser extent, in the neural retinas of AAV.GFP-treated eyes. Ultrastructurally, a reversal of RPE lipid droplet accumulation was observed at the AAV.RPE65 transgene injection site, but not at the site of injection of the control vector. CONCLUSIONS: In 10 of 11 treated RPE65(-/-) eyes, gene transfer resulted in development of vision, both subjectively apparent by loss of nystagmus, and objectively recorded by ERG. Structurally, there was reversal of lipid droplet accumulation in the RPE. Uveitis developed in 75% of the transgene-treated eyes, a complication possibly due to an immunopathogenic response to the RPE65 molecule.

Expression of beta-carotene 15,15' monooxygenase in retina and RPE-choroid.

PURPOSE: Beta-carotene 15,15' monooxygenase (beta-CM) catalyzes the central cleavage of beta-carotene to all-trans-retinal, the first step in vitamin A synthesis. This study was conducted to determine the expression of beta-CM in the mammalian retina and RPE, to assess its relevance in carotenoid-retinoid metabolism in the retina and RPE. METHODS: RT-PCR was used to detect expression of beta-CM mRNA in the retina and RPE-choroid of the mouse, cow, human, and monkey and in RPE cells and other cell lines. Immunofluorescence microscopy was used to localize beta-CM in mouse and monkey retina with an anti-peptide antibody specific for beta-CM. RESULTS: By RT-PCR, beta-CM mRNA was detected at a low level in mouse and monkey retina and in the RPE-choroid of the monkey but not of the mouse. Conversely, beta-CM mRNA was expressed at a low level in both human and bovine RPE-choroid, but not in the retina of either. RPE primary cultured cells of the monkey also showed beta-CM mRNA expression, although the three human lines did not. In addition, of nine other cell lines tested, only COS-7 was positive for beta-CM. Immunofluorescence microscopy showed weak immunoreactivity in the inner retina in both the mouse and monkey. beta-CM immunoreactivity was not detectable in RPE of the mouse. Use of a long-wavelength exciting and emitting secondary probe to mitigate lipofuscin autofluorescence, facilitated the detection of a low level of beta-CM immunoreactivity in monkey RPE. CONCLUSIONS: Beta-CM mRNA and protein are expressed at low levels in the mammalian retina and RPE-choroid. Given the low and variable expression of beta-CM in the retina and RPE, it can be concluded that beta-CM is not necessary for a conserved retina or RPE-specific function, but may be necessary for a species-specific function.

Expression and promoter activation of the Rpe65 gene in retinal pigment epithelium cell lines.

PURPOSE: To examine the expression and promoter activation of the retinal pigment epithelium (RPE)-preferentially expressed Rpe65 gene in the commonly available RPE cell lines. METHODS: Reverse transcription coupled to polymerase chain reaction (RT-PCR) was performed after total RNA extraction from different RPE (ARPE-19, monkey, hTERT-RP1 and D407) and non-RPE (COS-7, HeLa, HepG2 and HS27) cell lines. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the 5' flanking region of the mouse Rpe65 gene. The involvement of a putative TATA box in the basal promoter expression was studied by site-directed mutagenesis in D407 cells and binding of TATA box-related transcription factors to that region was demonstrated by Electrophoretic Mobility Shift Assays (EMSA). RESULTS: Expression of the human RPE65 cDNA was observed in all the RPE cell lines tested, and in COS-7 cells (monkey RPE65 cDNA). Transient transfections of the mouse Rpe65 promoter/luciferase transgene containing nested deletions of the Rpe65 5' flanking region showed that fragments containing bases -655 to +48 and -1240 to +48 generated specific promoter activity only in the D407 cell line. A promoter fragment from -49 to +48 directed basal promoter activity in all the cell lines tested. Part of this basal activity was due to a putative TATA box that specifically binds transcription factors contained in a D407 nuclear extract. CONCLUSIONS: Although transcription of the Rpe65 gene occurs in all the tested cell lines, we find that the D407 cell line is the only one capable of directing specific mouse Rpe65 promoter activity. This limits the study of the transcriptional regulation of the mouse Rpe65 gene in vitro to this particular cell line.

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-ß/BMP signaling and orphan nuclear receptors.

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of ß-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-ß signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-ß signaling and nuclear receptor) triggers axon retraction. We propose that a signal from a TGF-ß family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.

ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling.

Developmental axon pruning is a general mechanism that is required for maturation of neural circuits. During Drosophila metamorphosis, the larval-specific dendrites and axons of early γ neurons of the mushroom bodies are pruned and replaced by adult-specific processes. We found that the nuclear receptor ftz-f1 is required for this pruning, activates expression of the steroid hormone receptor EcR-B1, whose activity is essential for γ remodeling, and represses expression of Hr39, an ftz-f1 homologous gene. If inappropriately expressed in the γ neurons, HR39 inhibits normal pruning, probably by competing with endogenous FTZ-F1, which results in decreased EcR-B1 expression. EcR-B1 was previously identified as a target of the TGFß signaling pathway. We found that the ftz-f1 and Hr39 pathway apparently acts independently of TGFß signaling, suggesting that EcR-B1 is the target of two parallel molecular pathways that act during γ neuron remodeling.