Cyclin-G-associated kinase GAK/dAux regulates autophagy initiation via ULK1/Atg1 in glia.

Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinson's disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. Here, we present evidence that the PD risk factor, Cyclin-G-associated kinase (GAK)/Drosophila homolog Auxilin (dAux), is a component in glial autophagy. The lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally up-regulates levels of components in the initiation and PI3K class III complexes. GAK/dAux interacts with the master initiation regulator UNC-51like autophagy activating kinase 1/Atg1 via its uncoating domain and regulates the trafficking of Atg1 and Atg9 to autophagosomes, hence controlling the onset of glial autophagy. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles. Importantly, dAux contributes to PD-like symptoms including dopaminergic neurodegeneration and locomotor function in flies. Our findings identify an autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.

Synergistic action of 5Z-7-oxozeaenol and bortezomib in inducing apoptosis of Burkitt lymphoma cell line Daudi.

Treatment failure in cancer chemotherapy is largely due to the toxic effects of chemotherapeutic agents on normal cells/tissues. The proteasome inhibitor bortezomib has been successfully applied to treat multiple myeloma (MM), but there are some common adverse reactions in the clinic including peripheral neuropathy (PN). The TAK1 selective inhibitor 5Z-7-oxozeaenol has been widely studied in cancer therapy. Here, we investigated the potential synergy of bortezomib and 5Z-7-oxozeaenol in Burkitt's lymphoma (BL) cell lines. Cell viability assay showed that co-treatment of bortezomib at 8 nM, representing a one-eighth concentration for growth arrest, and 5Z-7-oxozeaenol at 2 µM, a dose that exhibited insignificant cytotoxic effects, synergistically induced apoptosis in the cell line Daudi. In parallel with the increasing dose of the bortezomib, and 5Z-7-oxozeaenol at 0.5 µM, lower colony formation efficiencies were seen in the cell line Daudi. Western blotting analysis verified that TAK1 inhibition by 5Z-7-oxozeaenol completely blocked JNK, p38, Erk, IKK, and IκB phosphorylation, which was almost instantly activated by TAK1 both directly or indirectly. Both agents synergistically prevented nuclear translocation of NF-κB, a characteristic of NF-κB inactivation. Moreover, a synergistic effect of bortezomib and 5Z-7-oxozeaenol on Western blotting analysis and flow cytometry was disclosed. Collectively, our results indicated that the proteasome inhibitor bortezomib and the TAK1 inhibitor 5Z-7-oxozeaenol displayed synergy on inhibiting BL cell apoptosis by inhibiting NF-κB activity.

Epithelial NELF guards intestinal barrier function to ameliorate colitis by maintaining junctional integrity.

Well-orchestrated transcriptional programs in intestinal epithelial cells (IECs) are essential for maintenance of optimal mucosal barrier functions, whereas the contribution of elongation-related mechanisms to barrier function remains unknown. Here, a combination of genetic and genomic approaches defined a critical role of IEC-intrinsic negative elongation factor (NELF) complex in maintenance of epithelial homeostasis. By direct occupancy at endogenous gene loci, NELF sustained expression of a subset of genes related to junctional integrity. As a result, epithelial NELF deficiency results in subdued levels of these junction-related genes and excessive IEC necroptosis in vivo secondary to commensal microbial invasion. In a colitis model, NELF-deficient mice exhibited severely impaired barrier integrity characterized by increased intestinal permeability and significantly exacerbated intestinal inflammation with lethal consequences. Our findings reveal the protective function of the NELF complex against intestinal damage and inflammation and suggest that elongation represents a biologically important step in defining IEC transcriptome.

α-Defensins Promote Bacteroides Colonization on Mucosal Reservoir to Prevent Antibiotic-Induced Dysbiosis.

In addition to their established functions in host defense, accumulating evidence has suggested an emerging role for antimicrobial proteins (AMPs) in shaping commensal microbiota. However, the role of α-defensins, the most abundant AMPs of intestine, in regulating microbial ecology remains inconclusive. Here, we report that α-defensins promote commensal Bacteroides colonization by enhancing bacterial adhesion to the mucosal reservoir. Experiments utilizing mice deficient in matrix metalloproteinase 7 (MMP7), the α-defensin-activating enzyme, with rigorous littermate controls showed that α-defensin deficiency did not significantly influence steady-state intestinal microbiota. In contrast, α-defensins are essential for replenishment of commensal Bacteroides from the mucosal reservoir following antibiotics-induced dysbiosis, shown by markedly compromised recovery of Bacteroides in Mmp7-/- animals. Mechanistically, α-defensins promote Bacteroides colonization on epithelial surfaces in vivo and adhesion to epithelial cells in vitro. Moreover, α-defensins unexpectedly does not show any microbicidal activities against Bacteroides. Together, we propose that α-defensins promote commensal bacterial colonization and recovery to maintain microbial diversity upon environmental challenges.

Nutrient Sensing by the Intestinal Epithelium Orchestrates Mucosal Antimicrobial Defense via Translational Control of Hes1.

Metabolic programs and host defense are highly integrated to ensure proper immune responses during stress. Central to these responses, mTOR regulates immune functions by sensing and integrating environmental cues, yet how these systems are coordinated at the intestinal surface remains undefined. We show that the antimicrobial peptide α-defensin is functionally sustained during nutrient deprivation because of regulation of the defensin-processing enzyme MMP7 by microbiota- and host-derived factors. Unlike other antimicrobial peptides, the MMP7-α-defensin axis remains active during nutrient fluctuations, providing essential protection against enteric pathogens. Sustained Mmp7 expression requires the microbiota and is mediated by de-repression of the transcription activator Atoh1 upon attenuation of the transcriptional repressor Hes1 in intestinal epithelial cells. Hes1 levels are regulated via mTOR and controlled translationally, constituting a metabolism-translation-transcription loop. Disrupting this loop by supplying nutrients paradoxically compromises antibacterial defense. Together, these results uncover a regulatory circuit that couples host nutrient status to epithelial antimicrobial immunity.

Auxilin Underlies Progressive Locomotor Deficits and Dopaminergic Neuron Loss in a Drosophila Model of Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disorder that exhibits motor and non-motor symptoms, as well as pathological hallmarks, including dopaminergic (DA) neuron death and formation of α-synuclein (α-Syn) Lewy bodies. Cyclin-G-associated kinase (GAK), a PD susceptibility gene identified through genome-wide association studies (GWAS), is a ubiquitous serine/threonine kinase involved in clathrin uncoating, though its PD-related function remains elusive. Here, we implicate the Drosophila GAK homolog, auxilin (aux), in a broad spectrum of parkinsonian-like symptoms. Downregulating aux expression leads to progressive loss of climbing ability, decreased lifespan, and age-dependent DA neuron death similar to α-Syn overexpression. Reduced aux expression further enhances and accelerates α-Syn-mediated DA neuron loss. Flies with reduced aux expression are more sensitive to the toxin paraquat, suggesting that genetic and environmental factors intertwine. Taken together, these findings decipher a pivotal role for GAK/aux and suggest mechanisms underlying PD.

Molecular Chaperone Calnexin Regulates the Function of Drosophila Sodium Channel Paralytic.

Neuronal activity mediated by voltage-gated channels provides the basis for higher-order behavioral tasks that orchestrate life. Chaperone-mediated regulation, one of the major means to control protein quality and function, is an essential route for controlling channel activity. Here we present evidence that Drosophila ER chaperone Calnexin colocalizes and interacts with the α subunit of sodium channel Paralytic. Co-immunoprecipitation analysis indicates that Calnexin interacts with Paralytic protein variants that contain glycosylation sites Asn313, 325, 343, 1463, and 1482. Downregulation of Calnexin expression results in a decrease in Paralytic protein levels, whereas overexpression of the Calnexin C-terminal calcium-binding domain triggers an increase reversely. Genetic analysis using adult climbing, seizure-induced paralysis, and neuromuscular junction indicates that lack of Calnexin expression enhances Paralytic-mediated locomotor deficits, suppresses Paralytic-mediated ghost bouton formation, and regulates minature excitatory junction potentials (mEJP) frequency and latency time. Taken together, our findings demonstrate a need for chaperone-mediated regulation on channel activity during locomotor control, providing the molecular basis for channlopathies such as epilepsy.

Analysis of Glial Distribution in Drosophila Adult Brains.

Neurons and glia are the two major cell types in the nervous system and work closely with each other to program neuronal interplay. Traditionally, neurons are thought to be the major cells that actively regulate processes like synapse formation, plasticity, and behavioral output. Glia, on the other hand, serve a more supporting role. To date, accumulating evidence has suggested that glia are active participants in virtually every aspect of neuronal function. Despite this, fundamental features of how glia interact with neurons, and their spatial relationships, remain elusive. Here, we describe the glial cell population in Drosophila adult brains. Glial cells extend and tightly associate their processes with major structures such as the mushroom body (MB), ellipsoid body (EB), and antennal lobe (AL) in the brain. Glial cells are distributed in a more concentrated manner in the MB. Furthermore, subsets of glia exhibit distinctive association patterns around different neuronal structures. Whereas processes extended by astrocyte-like glia and ensheathing glia wrap around the MB and infiltrate into the EB and AL, cortex glia stay where cell bodies of neurons are and remain outside of the synaptic regions structured by EB or AL.

Glial cells in neuronal development: recent advances and insights from Drosophila melanogaster.

Glia outnumber neurons and are the most abundant cell type in the nervous system. Whereas neurons are the major carriers, transducers, and processors of information, glial cells, once considered mainly to play a passive supporting role, are now recognized for their active contributions to almost every aspect of nervous system development. Recently, insights from the invertebrate organism Drosophila melanogaster have advanced our knowledge of glial cell biology. In particular, findings on neuron-glia interactions via intrinsic and extrinsic mechanisms have shed light on the importance of glia during different stages of neuronal development. Here, we summarize recent advances in understanding the functions of Drosophila glia, which resemble their mammalian counterparts in morphology and function, neural stem-cell conversion, synapse formation, and developmental axon pruning. These discoveries reinforce the idea that glia are substantial players in the developing nervous system and further advance the understanding of mechanisms leading to neurodegeneration.