The circular RNA Edis regulates neurodevelopment and innate immunity.

Circular RNAs (circRNAs) are widely expressed in eukaryotes. However, only a subset has been functionally characterized. We identify and validate a collection of circRNAs in Drosophila, and show that depletion of the brain-enriched circRNA Edis (circ_Ect4) causes hyperactivation of antibacterial innate immunity both in cultured cells and in vivo. Notably, Edis depleted flies display heightened resistance to bacterial infection and enhanced pathogen clearance. Conversely, ectopic Edis expression blocks innate immunity signaling. In addition, inactivation of Edis in vivo leads to impaired locomotor activity and shortened lifespan. Remarkably, these phenotypes can be recapitulated with neuron-specific depletion of Edis, accompanied by defective neurodevelopment. Furthermore, inactivation of Relish suppresses the innate immunity hyperactivation phenotype in the fly brain. Moreover, we provide evidence that Edis encodes a functional protein that associates with and compromises the processing and activation of the immune transcription factor Relish. Importantly, restoring Edis expression or ectopic expression of Edis-encoded protein suppresses both innate immunity and neurodevelopment phenotypes elicited by Edis depletion. Thus, our study establishes Edis as a key regulator of neurodevelopment and innate immunity.

Mask, the Drosophila ankyrin repeat and KH domain-containing protein, affects microtubule stability.

Proper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and remodeling in neurons. In the present study, we demonstrate that the Drosophila ankyrin repeat and KH domain-containing protein Mask negatively affects MT stability in both larval muscles and motor neurons. In larval muscles, loss-of-function of mask increases MT polymer length, and in motor neurons, loss of mask function results in overexpansion of the presynaptic terminal at the larval neuromuscular junctions (NMJs). mask genetically interacts with stathmin (stai), a neuronal modulator of MT stability, in the regulation of axon transportation and synaptic terminal stability. Our structure-function analysis of Mask revealed that its ankyrin repeats domain-containing N-terminal portion is sufficient to mediate Mask's impact on MT stability. Furthermore, we discovered that Mask negatively regulates the abundance of the MT-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss-of-function phenotypes at the larval NMJs. Taken together, our studies demonstrate that Mask is a novel regulator for MT stability, and such a role of Mask requires normal function of Jupiter.

Bilateral Transient Dilated and Fixed Pupils After Microvascular Decompression: Rare Clinical Experience.

Microvascular decompression (MVD) has a satisfactory safety, and it is the only surgical treatment for neurovascular compression diseases, such as hemifacial spasm, trigeminal neuralgia, and glossopharyngeal neuralgia, from the perspective of etiology. Bilateral dilated and fixed pupils have long been regarded as a sign of life threatening, which is common in patients with cerebral herniation due to cranial hypertension. However, transient dilated pupils after MVD have not been previously reported. Here, we presented 2 patients with bilateral transient dilated and fixed pupils after MVD and discussed the possible etiologies through the literature review. Physical examination of both patients showed bilateral pupils were normal and without a medical history of pupil dilation. They underwent MVD under general anesthesia and used propofol and sevoflurane. In both cases, the vertebral artery was displaced, and Teflon pads were inserted between the vertebral artery and the brain stem. Postoperation, we found transient bilateral mydriasis without light reflection in both patients. The emergency head computed tomography revealed no obvious signs of hemorrhage and cerebral herniation. About 1 hour later, this phenomenon disappeared. Therefore, the authors think if MVD is successfully carried out, bilateral transient mydriasis may not necessarily indicate brain stem hemorrhage, cerebral herniation, and other emergency conditions, which can be recovered within a short time. The causes could be related to stimulation of the sympathetic pathway in the brain stem during MVD and side effects of anesthetics.

Glossopharyngeal Neuralgia Characterized by Otalgia: A Retrospective Study.

Glossopharyngeal neuralgia (GPN) is an uncommon facial pain syndrome and is characterized by paroxysms of excruciating pain in the distributions of the auricular and pharyngeal branches of cranial nerves IX and X. Glossopharyngeal neuralgia characterized by otalgia alone is rare. Herein, the authors analyzed 2 patients with GPN with otalgia as the main clinical manifestation. The clinical features and prognosis of this rare group of patients with GPN were discussed. They both presented with paroxysmal pain in the external auditory meatus and preoperative magnetic resonance imaging suggested the vertebral artery were closely related to the glossopharyngeal nerves. In both patients, compression of the glossopharyngeal nerve was confirmed during microvascular decompression, and the symptoms were relieved immediately after surgery. At 11 to 15 months follow-up, there was no recurrence of pain. A variety of reasons can cause otalgia. The possibility of GPN is a clinical concern in patients with otalgia as the main complaint. The authors think the involvement of the glossopharyngeal nerve fibers in the tympanic plexus via Jacobson nerve may provide an important anatomic basis for GPN with predominant otalgia. Surface anesthesia test of the pharynx and preoperative magnetic resonance imaging is helpful for diagnosis. Microvascular decompression is effective in the treatment of GPN with predominant otalgia.

Self-assembly preparation of Zn2+ -immobilized silica hybrid monolith and application in solid-phase micro-extraction of ß-agonists.

Exploiting adsorbents with highly efficient extraction performance is of great promise for extracting small organic molecules from biological samples. In this work, a novel Zn2+ -immobilized chitosan@silica hybrid monolith was prepared through a simple self-assembly Zn2+ -immobilization process. Exploited as an adsorbent in solid-phase micro-extraction for extracting trace ß-agonists, the monolith exhibited high extraction efficiencies for salbutamol, clenbuterol, and ractopamine with the enrichment factors approaching 120, 85, and 52, respectively. These could be attributed to the effective interaction between Zn2+ ions and the target molecule via coordination or other intermolecular interactions. Under optimized extraction operations, a sensitive determination was successfully developed coupling with high-performance liquid chromatography-ultraviolet detection. The linear range was 0.17-58.8, 0.12-68.5, and 0.18-65.5 ng/ml for salbutamol, clenbuterol, and ractopamine. The limits of detection of the ß-agonists were from 0.04 to 0.07 ng/ml, and the limits of quantification were from 0.12 to 0.18 ng/ml. The recoveries of spiking in mutton samples were observed in the range of 85.9%-95.7%, with relative standard deviations <8.0% (n = 3). Application tests demonstrated this newly developed determination was practical, accurate, and convenient for detecting trace content ß-agonists in meat.

Comprehensive analysis and expression profiles of cassava UDP-glycosyltransferases (UGT) family reveal their involvement in development and stress responses in cassava.

UDP-glycosyltransferases (UGTs) are widely involved in plant growth and stress responses. However, UGT family are not well understood in cassava. Here, we identified 121 MeUGT genes and classified them into 14 subfamilies by phylogenetic analysis. All MeUGT proteins have typical feature of the UGTs family. Tandem duplications are the crucial driving force for the expansion of MeUGT family. Cis-Acting elements analysis uncovered those 14 kinds of cis-elements associated with biotic and abiotic stress responses. Transcriptomic and qRT-PCR analyses indicated that MeUGT genes participate in postharvest physiological deterioration of storage root and the responses of biotic and abiotic stresses. Of which, MeUGT-14/41 were significantly induced after Xam treatment. Silencing of MeUGT-14 or MeUGT-41 reduced cassava resistance to Xam, verifying the accuracy of transcriptomic data for function prediction. Together, this study characterized the MeUGTs family and revealed their potential functions, which build a solid foundation for MeUGTs associated genetic improvement of cassava.

Hemifacial Spasm Caused by Distal Neurovascular Compression Confirmed by Lateral Spread Response Monitoring.

Primary hemifacial spasm (HFS) is likely related to a vascular compression of the facial nerve at its distal cisternal portion root exit Zone that has been reported during recent years. Most of these cases were found during secondary surgery or intraoperative monitoring of lateral spread response (LSR). Here we reported 2 patients with typical HFS caused by distal neurovascular compression that were successfully treated with microvascular decompression. Magnetic resonance imaging in both cases suggested that there was a contact between the vessel in cisternal segment and the facial nerve. LSR immediately disappeared after decompression of distal neurovascular compression. Resolution of spasm after the operation was achieved in both of these cases, with a short duration of vertigo and mild facial paralysis in case 1. Reviewing the literature, the majority of cases of distal neurovascular compression are found under the following 2 conditions:(1) When patients underwent a second operation. (2) When surgeons explored the distal part, the cisternal portion, after exploring the traditional root exit Zone without LSR disappearing. Therefore, it is the distal neurovascular compression at cisternal segment that may also be the cause of HFS. As for this kind of special HFS, these patients may also present with cranial nerve symptoms of VIII. In addition, magnetic resonance imaging can provide some information about compression sites. When we perform microvascular decompression, we should carefully pay attention to having an entire-root-exploration with intraoperative electrophysiology to find and decompress the real neurovascular compression.

Highly dynamic, coordinated, and stage-specific profiles are revealed by a multi-omics integrative analysis during tuberous root development in cassava.

Cassava (Manihot esculenta) is an important starchy root crop that provides food for millions of people worldwide, but little is known about the regulation of the development of its tuberous root at the multi-omics level. In this study, the transcriptome, proteome, and metabolome were examined in parallel at seven time-points during the development of the tuberous root from the early to late stages of its growth. Overall, highly dynamic and stage-specific changes in the expression of genes/proteins were observed during development. Cell wall and auxin genes, which were regulated exclusively at the transcriptomic level, mainly functioned during the early stages. Starch biosynthesis, which was controlled at both the transcriptomic and proteomic levels, was mainly activated in the early stages and was greatly restricted during the late stages. Two main branches of lignin biosynthesis, coniferyl alcohol and sinapyl alcohol, also functioned during the early stages of development at both the transcriptomic and proteomic levels. Metabolomic analysis further supported the stage-specific roles of particular genes/proteins. Metabolites related to lignin and flavonoid biosynthesis showed high abundance during the early stages, those related to lipids exhibited high abundance at both the early and middle stages, while those related to amino acids were highly accumulated during the late stages. Our findings provide a comprehensive resource for broadening our understanding of tuberous root development and will facilitate future genetic improvement of cassava.

Genomic analyses of heat stress transcription factors (HSFs) in simulated drought stress response and storage root deterioration after harvest in cassava.

Heat shock factors (HSFs) play crucial roles in various plant stress responses. However, the current knowledge about HSFs in cassava, an important crop, is still insufficient. In this research, we identified 32 cassava HSF genes (MeHSFs) and clustered them into three groups (A, B, C) based on phylogenetic analysis and structural characteristics. Conserved motif analyses showed that MeHSFs display domains characteristic to HSF transcription factors. Gene structure analyses suggested that 29 MeHSFs contained only two exons. All identified 32 cassava MeHSFs were distributed on 13 chromosomes. Their expression profiles revealed that the different MeHSFs were expressed differentially in different tissues, most high expression genes belonged to group A. The similar MeHSFs were up-regulated after treatment with both PEG and abscisic acid (ABA), which implied that these MeHSFs may participate in resistance to simulated drought stress associated with the ABA signaling pathway. In addition, several MeHSFs were induced during postharvest physiological deterioration (PPD) in cassava. Our results provided basic but important knowledge for future gene function analysis of MeHSFs toward efforts in improving tolerance to abiotic stress and PPD in cassava.

Strand-specific RNA-seq based identification and functional prediction of drought-responsive lncRNAs in cassava.

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as playing crucial roles in abiotic stress responsive regulation, however, the mechanism of lncRNAs underlying drought-tolerance remains largely unknown in cassava, an important tropical and sub-tropical root crop of remarkable drought tolerance. RESULTS: In this study, a total of 833 high-confidence lncRNAs, including 652 intergenic and 181 anti-sense lncRNAs, were identified in cassava leaves and root using strand-specific RNA-seq technology, of which 124 were drought-responsive. Trans-regulatory co-expression network revealed that lncRNAs exhibited tissue-specific expression patterns and they preferred to function differently in distinct tissues: e.g., cell-related metabolism, cell wall, and RNA regulation of transcription in folded leaf (FL); degradation of major carbohydrate (CHO) metabolism, calvin cycle and light reaction, light signaling, and tetrapyrrole synthesis in full expanded leaf (FEL); synthesis of major CHO metabolism, nitrogen-metabolism, photosynthesis, and redox in bottom leaf (BL); and hormone metabolism, secondary metabolism, calcium signaling, and abiotic stress in root (RT). In addition, 27 lncRNA-mRNA pairs referred to cis-acting regulation were identified, and these lncRNAs regulated the expression of their neighboring genes mainly through hormone metabolism, RNA regulation of transcription, and signaling of receptor kinase. Besides, 11 lncRNAs were identified acting as putative target mimics of known miRNAs in cassava. Finally, five drought-responsive lncRNAs and 13 co-expressed genes involved in trans-acting, cis-acting, or target mimic regulation were selected and confirmed by qRT-PCR. CONCLUSIONS: These findings provide a comprehensive view of cassava lncRNAs in response to drought stress, which will enable in-depth functional analysis in the future.

Genome-wide identification and expression profiling of glutathione transferase gene family under multiple stresses and hormone treatments in wheat (Triticum aestivum L.).

BACKGROUND: Glutathione transferases (GSTs), the ancient, ubiquitous and multi-functional proteins, play significant roles in development, metabolism as well as abiotic and biotic stress responses in plants. Wheat is one of the most important crops, but the functions of GST genes in wheat were less studied. RESULTS: A total of 330 TaGST genes were identified from the wheat genome and named according to the nomenclature of rice and Arabidopsis GST genes. They were classified into eight classes based on the phylogenetic relationship among wheat, rice, and Arabidopsis, and their gene structure and conserved motif were similar in the same phylogenetic class. The 43 and 171 gene pairs were identified as tandem and segmental duplication genes respectively, and the Ka/Ks ratios of tandem and segmental duplication TaGST genes were less than 1 except segmental duplication gene pair TaGSTU24/TaGSTU154. The 59 TaGST genes were identified to have syntenic relationships with 28 OsGST genes. The expression profiling involved in 15 tissues and biotic and abiotic stresses suggested the different expression and response patterns of the TaGST genes. Furthermore, the qRT-PCR data showed that GST could response to abiotic stresses and hormones extensively in wheat. CONCLUSIONS: In this study, a large GST family with 330 members was identified from the wheat genome. Duplication events containing tandem and segmental duplication contributed to the expansion of TaGST family, and duplication genes might undergo extensive purifying selection. The expression profiling and cis-elements in promoter region of 330 TaGST genes implied their roles in growth and development as well as adaption to stressful environments. The qRT-PCR data of 14 TaGST genes revealed that they could respond to different abiotic stresses and hormones, especially salt stress and abscisic acid. In conclusion, this study contributed to the further functional analysis of GST genes family in wheat.

Cranioplasty Using Polymethylmethacrylate Cement Following Retrosigmoid Craniectomy Decreases the Rate of Cerebrospinal Fluid Leak and Pseudomeningocele.

OBJECTIVE: Cerebrospinal fluid (CSF) leak frequently occurs after retrosigmoid craniectomy. The present study investigated the effects of cranioplasty using polymethylmethacrylate (PMMA) cement to reduce the incidence of CSF leak following retrosigmoid craniectomy as compared with the autologous bone flap combined with titanium plates. METHODS: Two hundred forty-three patients underwent surgeries via retrosigmoid approach for microvascular decompression or tumor resection. Of these, 107 patients underwent craniotomy, and incomplete cranioplasty was performed with autologous bone flap fixed with titanium plates, while 136 patients underwent craniectomy and complete cranioplasty was performed with PMMA cement. Variables including the incidence of CSF leak, pseudomeningocele formation, wound infection, rejection reaction were compared retrospectively based on the clinical data between the 2 groups. RESULTS: In the autologous bone group, 9 patients had postoperative CSF leaks, and 11 patients had pseudomeningoceles, while 3 CSF leaks and 2 pseudomeningoceles were found in the PMMA group. Statistical analysis showed that PMMA significantly decreased the incidence of postoperative CSF leaks (P = 0.03) and pseudomeningocele formation (P = 0.002). Wound infections were observed in 2 and 1 patients between the autologous bone and PMMA group, respectively, which did not differ significantly (P = 0.58). None of the patients in both groups developed a rejection reaction of artificial materials. CONCLUSIONS: Complete cranioplasty with PMMA cement following retrosigmoid craniectomy could decrease the incidence of CSF leak and pseudomeningocele formation as compared with the autologous bone flap combined with titanium plates. Thus, PMMA cement is preferable for bone reconstruction with excellent biocompatibility and without increasing the rate of wound infection.

The Class III Peroxidase (POD) Gene Family in Cassava: Identification, Phylogeny, Duplication, and Expression.

The class III peroxidase (POD) enzymes participate in plant development, hormone signaling, and stress responses. However, little is known about the POD family in cassava. Here, we identified 91 cassava POD genes (MePODs) and classified them into six subgroups using phylogenetic analysis. Conserved motif analysis demonstrated that all MePOD proteins have typical peroxidase domains, and gene structure analysis showed that MePOD genes have between one and nine exons. Duplication pattern analysis suggests that tandem duplication has played a role in MePOD gene expansion. Comprehensive transcriptomic analysis revealed that MePOD genes in cassava are involved in the drought response and postharvest physiological deterioration. Several MePODs underwent transcriptional changes after various stresses and related signaling treatments were applied. In sum, we characterized the POD family in cassava and uncovered the transcriptional control of POD genes in response to various stresses and postharvest physiological deterioration conditions. These results can be used to identify potential target genes for improving the stress tolerance of cassava crops.

The Late Embryogenesis Abundant Protein Family in Cassava (Manihot esculenta Crantz): Genome-Wide Characterization and Expression during Abiotic Stress.

Late embryogenesis abundant (LEA) proteins, as a highly diverse group of polypeptides, play an important role in plant adaptation to abiotic stress; however, LEAs from cassava have not been studied in cassava. In this study, 26 LEA members were genome-wide identified from cassava, which were clustered into seven subfamily according to evolutionary relationship, protein motif, and gene structure analyses. Chromosomal location and duplication event analyses suggested that 26 MeLEAs distributed in 10 chromosomes and 11 MeLEA paralogues were subjected to purifying selection. Transcriptomic analysis showed the expression profiles of MeLEAs in different tissues of stem, leaves, and storage roots of three accessions. Comparative transcriptomic analysis revealed that the function of MeLEAs in response to drought may be differentiated in different accessions. Compared with the wild subspecies W14, more MeLEA genes were activated in cultivated varieties Arg7 and SC124 after drought treatment. Several MeLEA genes showed induction under various stresses and related signaling treatments. Taken together, this study demonstrates the transcriptional control of MeLEAs in tissue development and the responses to abiotic stress in cassava and identifies candidate genes for improving crop resistance to abiotic stress.

Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants.

PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. Here, we show that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. We found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. We also provide evidence that loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, our data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions.

The core regulatory network of the abscisic acid pathway in banana: genome-wide identification and expression analyses during development, ripening, and abiotic stress.

BACKGROUND: Abscisic acid (ABA) signaling plays a crucial role in developmental and environmental adaptation processes of plants. However, the PYL-PP2C-SnRK2 families that function as the core components of ABA signaling are not well understood in banana. RESULTS: In the present study, 24 PYL, 87 PP2C, and 11 SnRK2 genes were identified from banana, which was further supported by evolutionary relationships, conserved motif and gene structure analyses. The comprehensive transcriptomic analyses showed that banana PYL-PP2C-SnRK2 genes are involved in tissue development, fruit development and ripening, and response to abiotic stress in two cultivated varieties. Moreover, comparative expression analyses of PYL-PP2C-SnRK2 genes between BaXi Jiao (BX) and Fen Jiao (FJ) revealed that PYL-PP2C-SnRK2-mediated ABA signaling might positively regulate banana fruit ripening and tolerance to cold, salt, and osmotic stresses. Finally, interaction networks and co-expression assays demonstrated that the core components of ABA signaling were more active in FJ than in BX in response to abiotic stress, further supporting the crucial role of the genes in tolerance to abiotic stress in banana. CONCLUSIONS: This study provides new insights into the complicated transcriptional control of PYL-PP2C-SnRK2 genes, improves the understanding of PYL-PP2C-SnRK2-mediated ABA signaling in the regulation of fruit development, ripening, and response to abiotic stress, and identifies some candidate genes for genetic improvement of banana.

A Cullin1-based SCF E3 ubiquitin ligase targets the InR/PI3K/TOR pathway to regulate neuronal pruning.

Pruning that selectively eliminates unnecessary axons/dendrites is crucial for sculpting the nervous system during development. During Drosophila metamorphosis, dendrite arborization neurons, ddaCs, selectively prune their larval dendrites in response to the steroid hormone ecdysone, whereas mushroom body γ neurons specifically eliminate their axon branches within dorsal and medial lobes. However, it is unknown which E3 ligase directs these two modes of pruning. Here, we identified a conserved SCF E3 ubiquitin ligase that plays a critical role in pruning of both ddaC dendrites and mushroom body γ axons. The SCF E3 ligase consists of four core components Cullin1/Roc1a/SkpA/Slimb and promotes ddaC dendrite pruning downstream of EcR-B1 and Sox14, but independently of Mical. Moreover, we demonstrate that the Cullin1-based E3 ligase facilitates ddaC dendrite pruning primarily through inactivation of the InR/PI3K/TOR pathway. We show that the F-box protein Slimb forms a complex with Akt, an activator of the InR/PI3K/TOR pathway, and promotes Akt ubiquitination. Activation of the InR/PI3K/TOR pathway is sufficient to inhibit ddaC dendrite pruning. Thus, our findings provide a novel link between the E3 ligase and the InR/PI3K/TOR pathway during dendrite pruning.

SkpA restrains synaptic terminal growth during development and promotes axonal degeneration following injury.

The Wallenda (Wnd)/dual leucine zipper kinase (DLK)-Jnk pathway is an evolutionarily conserved MAPK signaling pathway that functions during neuronal development and following axonal injury. Improper pathway activation causes defects in axonal guidance and synaptic growth, whereas loss-of-function mutations in pathway components impairs axonal regeneration and degeneration after injury. Regulation of this pathway is in part through the E3 ubiquitin ligase Highwire (Hiw), which targets Wnd/DLK for degradation to limit MAPK signaling. To explore mechanisms controlling Wnd/DLK signaling, we performed a large-scale genetic screen in Drosophila to identify negative regulators of the pathway. Here we describe the identification and characterization of SkpA, a core component of SCF E3 ubiquitin ligases. Mutants in SkpA display synaptic overgrowth and an increase in Jnk signaling, similar to hiw mutants. The combination of hypomorphic alleles of SkpA and hiw leads to enhanced synaptic growth. Mutants in the Wnd-Jnk pathway suppress the overgrowth of SkpA mutants demonstrating that the synaptic overgrowth is due to increased Jnk signaling. These findings support the model that SkpA and the E3 ligase Hiw function as part of an SCF-like complex that attenuates Wnd/DLK signaling. In addition, SkpA, like Hiw, is required for synaptic and axonal responses to injury. Synapses in SkpA mutants are more stable following genetic or traumatic axonal injury, and axon loss is delayed in SkpA mutants after nerve crush. As in highwire mutants, this axonal protection requires Nmnat. Hence, SkpA is a novel negative regulator of the Wnd-Jnk pathway that functions with Hiw to regulate both synaptic development and axonal maintenance.

Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons.

During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B' [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3ß kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot).

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.