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.

A LRRK2/dLRRK-mediated lysosomal pathway that contributes to glial cell death and DA neuron survival.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease. A plethora of evidence has indicated a role for LRRK2 in endolysosomal trafficking in neurons, while LRRK2 function in glia, although highly expressed, remains largely unknown. Here, we present evidence that LRRK2/dLRRK mediates a lysosomal pathway that contributes to glial cell death and the survival of dopaminergic (DA) neurons. LRRK2/dLRRK knockdown in the immortalized microglia or flies results in enlarged and swelling lysosomes fewer in number. These lysosomes are less mobile, wrongly acidified, exhibit defective membrane permeability and reduced activity of the lysosome hydrolase cathepsin B. In addition, LRRK2/dLRRK depletion causes glial apoptosis, DA neurodegeneration, and locomotor deficits in an age-dependent manner. Taken together, these findings demonstrate a functional role of LRRK2/dLRRK in regulating the glial lysosomal pathway; deficits in lysosomal biogenesis and function linking to glial apoptosis potentially underlie the mechanism of DA neurodegeneration, providing insights on LRRK2/dLRRK function in normal and pathological brains.

Glia-derived temporal signals orchestrate neurogenesis in the Drosophila mushroom body.

Intrinsic mechanisms such as temporal series of transcription factors orchestrate neurogenesis from a limited number of neural progenitors in the brain. Extrinsic regulations, however, remain largely unexplored. Here we describe a two-step glia-derived signal that regulates neurogenesis in the Drosophila mushroom body (MB). In a temporal manner, glial-specific ubiquitin ligase dSmurf activates non-cell-autonomous Hedgehog signaling propagation by targeting the receptor Patched to suppress and promote the exit of MB neuroblast (NB) proliferation, thereby specifying the correct α/ß cell number without affecting differentiation. Independent of NB proliferation, dSmurf also stabilizes the expression of the cell-adhesion molecule Fasciclin II (FasII) via its WW domains and regulates FasII homophilic interaction between glia and MB axons to refine α/ß-lobe integrity. Our findings provide insights into how extrinsic glia-to-neuron communication coordinates with NB proliferation capacity to regulate MB neurogenesis; glial proteostasis is likely a generalized mechanism in orchestrating neurogenesis.

Pathogenesis of α-Synuclein in Parkinson's Disease: From a Neuron-Glia Crosstalk Perspective.

Parkinson's disease (PD) is a progressive neurodegenerative disorder. The classical behavioral defects of PD patients involve motor symptoms such as bradykinesia, tremor, and rigidity, as well as non-motor symptoms such as anosmia, depression, and cognitive impairment. Pathologically, the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN) and the accumulation of α-synuclein (α-syn)-composed Lewy bodies (LBs) and Lewy neurites (LNs) are key hallmarks. Glia are more than mere bystanders that simply support neurons, they actively contribute to almost every aspect of neuronal development and function; glial dysregulation has been implicated in a series of neurodegenerative diseases including PD. Importantly, amounting evidence has added glial activation and neuroinflammation as new features of PD onset and progression. Thus, gaining a better understanding of glia, especially neuron-glia crosstalk, will not only provide insight into brain physiology events but also advance our knowledge of PD pathologies. This review addresses the current understanding of α-syn pathogenesis in PD, with a focus on neuron-glia crosstalk. Particularly, the transmission of α-syn between neurons and glia, α-syn-induced glial activation, and feedbacks of glial activation on DA neuron degeneration are thoroughly discussed. In addition, α-syn aggregation, iron deposition, and glial activation in regulating DA neuron ferroptosis in PD are covered. Lastly, we summarize the preclinical and clinical therapies, especially targeting glia, in PD treatments.

The TEA domain family transcription factor TEAD4 represses murine adipogenesis by recruiting the cofactors VGLL4 and CtBP2 into a transcriptional complex.

The Hippo signaling pathway is known to play an important role in multiple physiological processes, including adipogenesis. However, whether the downstream components of the Hippo pathway are involved in adipogenesis remains unknown. Here we demonstrate that the TEA domain family (TEAD) transcription factors are essential for adipogenesis in murine 3T3-L1 preadipocytes. Knockdown of TEAD1-4 stimulated adipogenesis and increased the expression of adipocyte markers in these cells. Interestingly, we found that the TEAD4 knockdown-mediated adipogenesis proceeded in a Yes-associated protein (YAP)/TAZ (Wwtr1)-independent manner and that adipogenesis suppression in WT cells involved formation of a ternary complex comprising TEAD4 and the transcriptional cofactors C-terminal binding protein 2 (CtBP2) and vestigial-like family member 4 (VGLL4). VGLL4 acted as an adaptor protein that enhanced the interaction between TEAD4 and CtBP2, and this TEAD4-VGLL4-CtBP2 ternary complex dynamically existed at the early stage of adipogenesis. Finally, we verified that TEAD4 directly targets the promoters of major adipogenesis transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and adiponectin, C1Q, and collagen domain-containing (Adipoq) during adipogenesis. These findings reveal critical insights into the role of the TEAD4-VGLL4-CtBP2 transcriptional repressor complex in suppression of adipogenesis in murine preadipocytes.

Microglia in Parkinson's Disease.

Microglia are the most abundant immune cells in the central nervous system (CNS), where they interact with neurons and exhibit a wide array of functions in physiological and pathological conditions. Physiologically, microglia mediate synaptic pruning and remodeling crucial for neural circuits and brain connectivity. In pathological conditions such as neurodegeneration in the Parkinson's disease (PD), microglia are activated, migrated to the injury site, and prone to engulf debris, sense pathology, and secrete possible pro- and anti-inflammatory factors. Microglia mediate responses such as inflammation and phagocytosis associated with neurodegeneration and are pivotal players in exacerbating or relieving disease progression. This chapter provides an overview on microglial function in the neurodegenerative disease-Parkinson's disease (PD). An overview on the pathology of PD will first be given, followed by discussion on receptors and signaling pathways involved in microglia-mediated inflammation and phagocytosis. Mechanism of how microglia contribute to PD by inflammation, phagocytosis of α-Synuclein (α-Syn), and interaction with PD genes will also be discussed.

Evolution of Neuroglia.

As the nervous system evolved from the diffused to centralised form, the neurones were joined by the appearance of the supportive cells, the neuroglia. Arguably, these non-neuronal cells evolve into a more diversified cell family than the neurones are. The first ancestral neuroglia appeared in flatworms being mesenchymal in origin. In the nematode C. elegans proto-astrocytes/supportive glia of ectodermal origin emerged, albeit the ensheathment of axons by glial cells occurred later in prawns. The multilayered myelin occurred by convergent evolution of oligodendrocytes and Schwann cells in vertebrates above the jawless fishes. Nutritive partitioning of the brain from the rest of the body appeared in insects when the hemolymph-brain barrier, a predecessor of the blood-brain barrier was formed. The defensive cellular mechanism required specialisation of bona fide immune cells, microglia, a process that occurred in the nervous system of leeches, bivalves, snails, insects and above. In ascending phylogeny, new type of glial cells, such as scaffolding radial glia, appeared and as the bran sizes enlarged, the glia to neurone ratio increased. Humans possess some unique glial cells not seen in other animals.

The Concept of Neuroglia.

Neuroglia represent a diverse population of non-neuronal cells in the nervous systems, be that peripheral, central, enteric or autonomic nervous system. Arguably, these cells represent about half of the volume of the human brain. This volumetric ratio, and by extension glia to neurone ratio, not only widely differ depending on the size of the animal species brain and its positioning on the phylogenetic tree, but also vary between the regions of an individual brain. Neuroglia derived from a dual origin (ectoderm and mesodermal) and in an assorted morphology, yet their functional traits can be mainly classified into being keepers of homeostasis (water, ions, neurotransmitters, metabolites, fuels, etc.) and defenders (e.g., against microbial organisms, etc.) of the nervous system. As these capabilities go awry, neuroglia ultimately define their fundamental role in most, if not, all neuropathologies. This concept presented in this chapter serves as a general introduction into the world of neuroglia and subsequent topics covered by this book.

General Pathophysiology of Astroglia.

Astroglial cells are involved in most if not in all pathologies of the brain. These cells can change the morpho-functional properties in response to pathology or innate changes of these cells can lead to pathologies. Overall pathological changes in astroglia are complex and diverse and often vary with different disease stages. We classify astrogliopathologies into reactive astrogliosis, astrodegeneration with astroglial atrophy and loss of function, and pathological remodelling of astrocytes. Such changes can occur in neurological, neurodevelopmental, metabolic and psychiatric disorders as well as in infection and toxic insults. Mutation in astrocyte-specific genes leads to specific pathologies, such as Alexander disease, which is a leukodystrophy. We discuss changes in astroglia in the pathological context and identify some molecular entities underlying pathology. These entities within astroglia may repent targets for novel therapeutic intervention in the management of brain pathologies.

A new regulator of autophagy initiation in glia.

Macroautophagy/autophagy is the major degradation pathway in neurons for eliminating damaged proteins and organelles in Parkinson disease (PD). Like neurons, glial cells are important contributors to PD, yet how autophagy is executed in glia and whether it is using similar interplay as in neurons or other tissues, remain largely elusive. Recently, we reported that the PD risk factor, GAK/aux (cyclin-G-associated kinase/auxilin), regulates the onset of glial autophagy. In the absence of GAK/aux, the number and size of the autophagosomes and autophagosomal precursors increase in adult fly glia and mouse microglia. The protein levels of components in the initiation and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes are generally upregulated. GAK/aux interacts with the master initiation regulator ULK1/Atg1 (unc-51 like autophagy activating kinase 1) via its uncoating domain, hinders autophagy activation by competing with ATG13 (autophagy related 13) for binding to the ULK1 C terminus, and regulates ULK1 trafficking to phagophores. Nonetheless, lack of GAK/aux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/aux might play additional roles. Overall, our findings reveal a new regulator of autophagy initiation in glia, advancing our understanding on how glia contribute to PD in terms of eliminating pathological protein aggregates.Abbreviations: ATG13: autophagy related 13; GAK/aux: cyclin G associated kinase/auxilin; PtdIns3K: phosphatidylinositol 3-kinase; PD: Parkinson disease; ULK1/Atg1: unc-51 like autophagy activating kinase 1.

The autophagy protein Atg9 functions in glia and contributes to parkinsonian symptoms in a Drosophila model of Parkinson's disease.

Parkinson's disease is a progressive neurodegenerative disease characterized by motor deficits, dopaminergic neuron loss, and brain accumulation of α-synuclein aggregates called Lewy bodies. Dysfunction in protein degradation pathways, such as autophagy, has been demonstrated in neurons as a critical mechanism for eliminating protein aggregates in Parkinson's disease. However, it is less well understood how protein aggregates are eliminated in glia, the other cell type in the brain. In the present study, we show that autophagy-related gene 9 (Atg9), the only transmembrane protein in the autophagy machinery, is highly expressed in Drosophila glia from adult brain. Results from immunostaining and live cell imaging analysis reveal that a portion of Atg9 localizes to the trans-Golgi network, autophagosomes, and lysosomes in glia. Atg9 is persistently in contact with these organelles. Lacking glial atg9 reduces the number of omegasomes and autophagosomes, and impairs autophagic substrate degradation. This suggests that glial Atg9 participates in the early steps of autophagy, and hence the control of autophagic degradation. Importantly, loss of glial atg9 induces parkinsonian symptoms in Drosophila including progressive loss of dopaminergic neurons, locomotion deficits, and glial activation. Our findings identify a functional role of Atg9 in glial autophagy and establish a potential link between glial autophagy and Parkinson's disease. These results may provide new insights on the underlying mechanism of Parkinson's disease.

Gcm proteins function in the developing nervous system.

A fundamental issue during nervous system development is how individual cells are formed from the undefined precursors. Differentiated neurons and glia, two major cell types mediating neuronal function, are acquired from immature precursors via a series of explicit controls exerted by transcription factors such as proteins in the family of Glial cells missing (Gcm). In mammals, Gcm proteins are involved in placenta and parathyroid gland development, whereas in the invertebrate organism Drosophila, Gcm proteins act as fate determinants for glial cell fate, regulate neural stem cell (NSC) induction and conversion, and promote glial proliferation. In particular, Gcm protein levels are carefully tuned for Drosophila gliogenesis and their stability is under precise control via the ubiquitin-proteasome system (UPS). Here we summarize recent advances on Gcm proteins function. In addition to describe various features of Gcm protein family, the significance of their functions in the developing nervous system is also discussed.

Profiling neurotransmitter-evoked glial responses by RNA-sequencing analysis.

Fundamental properties of neurons and glia are distinctively different. Neurons are excitable cells that transmit information, whereas glia have long been considered as passive bystanders. Recently, the concept of tripartite synapse is proposed that glia are structurally and functionally incorporated into the synapse, the basic unit of information processing in the brains. It has then become intriguing how glia actively communicate with the presynaptic and postsynaptic compartments to influence the signal transmission. Here we present a thorough analysis at the transcriptional level on how glia respond to different types of neurotransmitters. Adult fly glia were purified from brains incubated with different types of neurotransmitters ex vivo. Subsequent RNA-sequencing analyses reveal distinct and overlapping patterns for these transcriptomes. Whereas Acetylcholine (ACh) and Glutamate (Glu) more vigorously activate glial gene expression, GABA retains its inhibitory effect. All neurotransmitters fail to trigger a significant change in the expression of their synthesis enzymes, yet Glu triggers increased expression of neurotransmitter receptors including its own and nAChRs. Expressions of transporters for GABA and Glutamate are under diverse controls from DA, GABA, and Glu, suggesting that the evoked intracellular pathways by these neurotransmitters are interconnected. Furthermore, changes in the expression of genes involved in calcium signaling also functionally predict the change in the glial activity. Finally, neurotransmitters also trigger a general metabolic suppression in glia except the DA, which upregulates a number of genes involved in transporting nutrients and amino acids. Our findings fundamentally dissect the transcriptional change in glia facing neuronal challenges; these results provide insights on how glia and neurons crosstalk in a synaptic context and underlie the mechanism of brain function and behavior.

Antioxidant Blend of Curcumin and Broccoli Seed Extract Exhibits Protective Effect on Neurodegeneration and Promotes Drosophila Lifespan.

Antioxidants and related compounds are anti-inflammatory and exhibit great potential in promoting human health. They are also often considered to be important elements in the process of neurodegeneration. Here we describe a antioxidant blend of Curcumin and Broccoli Seed Extract (BSE). Flies treated with the blend exhibit extended lifespan. RNA-seq analysis of samples from adult fly brains reveals a wide array of new genes with differential expression upon treatment with the blend. Interestingly, abolishing expression of some of the identified genes in dopaminergic (DA) neurons does not affect DA neuron number. Taken together, our findings reveal an antioxidant blend that promotes fly longevity and exhibits protective effect over neurodegeneration, demonstrating the importance of antioxidants in health and pathology.

The noncanonical role of the protease cathepsin D as a cofilin phosphatase.

Cathepsin D (cathD) is traditionally regarded as a lysosomal protease that degrades substrates in acidic compartments. Here we report cathD plays an unconventional role as a cofilin phosphatase orchestrating actin remodeling. In neutral pH environments, the cathD precursor directly dephosphorylates and activates the actin-severing protein cofilin independent of its proteolytic activity, whereas mature cathD degrades cofilin in acidic pH conditions. During development, cathD complements the canonical cofilin phosphatase slingshot and regulates the morphogenesis of actin-based structures. Moreover, suppression of cathD phosphatase activity leads to defective actin organization and cytokinesis failure. Our findings identify cathD as a dual-function molecule, whose functional switch is regulated by environmental pH and its maturation state, and reveal a novel regulatory role of cathD in actin-based cellular processes.

Late endosomes promote microglia migration via cytosolic translocation of immature protease cathD.

Organelle transport requires dynamic cytoskeleton remodeling, but whether cytoskeletal dynamics are, in turn, regulated by organelles remains elusive. Here, we demonstrate that late endosomes, a type of prelysosomal organelles, facilitate actin-cytoskeleton remodeling via cytosolic translocation of immature protease cathepsin D (cathD) during microglia migration. After cytosolic translocation, late endosome-derived cathD juxtaposes actin filaments at the leading edge of lamellipodia. Suppressing cathD expression or blocking its cytosolic translocation impairs the maintenance but not the initiation of lamellipodial extension. Moreover, immature cathD balances the activity of the actin-severing protein cofilin to maintain globular-actin (G-actin) monomer pool for local actin recycling. Our study identifies cathD as a key lysosomal molecule that unconventionally contributes to actin cytoskeleton remodeling via cytosolic translocation during adenosine triphosphate-evoked microglia migration.

Lola regulates Drosophila adult midgut homeostasis via non-canonical hippo signaling.

Tissue homeostasis and regeneration in the Drosophila midgut is regulated by a diverse array of signaling pathways including the Hippo pathway. Hippo signaling restricts intestinal stem cell (ISC) proliferation by sequestering the transcription co-factor Yorkie (Yki) in the cytoplasm, a factor required for rapid ISC proliferation under injury-induced regeneration. Nonetheless, the mechanism of Hippo-mediated midgut homeostasis and whether canonical Hippo signaling is involved in ISC basal proliferation are less characterized. Here we identify Lola as a transcription factor acting downstream of Hippo signaling to restrict ISC proliferation in a Yki-independent manner. Not only that Lola interacts with and is stabilized by the Hippo signaling core kinase Warts (Wts), Lola rescues the enhanced ISC proliferation upon Wts depletion via suppressing Dref and SkpA expressions. Our findings reveal that Lola is a non-canonical Hippo signaling component in regulating midgut homeostasis, providing insights on the mechanism of tissue maintenance and intestinal function.

Traffic jam regulates the function of the ovarian germline stem cell progeny differentiation niche during pre-adult stage in Drosophila.

Stem cell self-renewal and the daughter cell differentiation are tightly regulated by the respective niches, which produce extrinsic cues to support the proper development. In Drosophila ovary, Dpp is secreted from germline stem cell (GSC) niche and activates the BMP signaling in GSCs for their self-renewal. Escort cells (ECs) in differentiation niche restrict Dpp outside the GSC niche and extend protrusions to help with proper differentiation of the GSC daughter cells. Here we provide evidence that loss of large Maf transcriptional factor Traffic jam (Tj) blocks GSC progeny differentiation. Spatio-temporal specific knockdown experiments indicate that Tj is required in pre-adult EC lineage for germline differentiation control. Further molecular and genetic analyses suggest that the defective germline differentiation caused by tj-depletion is partly attributed to the elevated dpp in the differentiation niche. Moreover, our study reveals that tj-depletion induces ectopic En expression outside the GSC niche, which contributes to the upregulated dpp expression in ECs as well as GSC progeny differentiation defect. Alternatively, loss of EC protrusions and decreased EC number elicited by tj-depletion may also partially contribute to the germline differentiation defect. Collectively, our findings suggest that Tj in ECs regulates germline differentiation by controlling the differentiation niche characteristics.

Wingless modulates activator protein-1-mediated tumor invasion.

Metastasis begins with a subset of local tumor cells acquiring the potential to invade into surrounding tissues, and remains to be a major obstacle for cancer treatments. More than 90% of cancer patients died from tumor metastasis, instead of primary tumor growth. The canonical Wnt/ß-catenin pathway plays essential roles in promoting tumor formation, yet its function in regulating tumor metastasis and the underlying mechanisms remain controversial. Here we employed well-established Drosophila tumor models to investigate the regulating mechanism of Wingless (Wg) pathway in tumor invasion. Our results showed that Wg signaling is necessary and sufficient for cell polarity disruption-induced cell migration and molecular changes reminiscent of epithelial-mesenchymal transition (EMT). Moreover, reducing Wg signaling suppressed lgl-/-/RasV12-induced tumor invasion, and cooperation between Arm and RasV12 is sufficient to induce tumor invasion. Mechanistically, we found that cell polarity disruption activates JNK signaling, which in turn upregulate wg expression through transcription factor activator protein-1 (AP-1). We identified a consensus AP-1 binding site located in the 2nd intron of wg, and confirmed that it is essential for AP-1 induced wg transcription both in vitro and in vivo. Lastly, we confirmed that the transcriptional activation of WNT by AP-1 is conserved in human cancer cells. These evidences reveal a positive role of Wnt/ß-catenin pathway in tumor invasion, and provide a conserved mechanism that connects JNK and Wnt signaling in regulating tumor progression.

Prevalence of structural central nervous system abnormalities in early-onset type 1 diabetes mellitus.

OBJECTIVE: To characterize the effects of severe hypoglycemia on the developing brain in children with early-onset type 1 diabetes mellitus (T1DM). STUDY DESIGN: Children diagnosed with T1DM before age 6 years were studied. Those with prospectively monitored severe hypoglycemia (coma/seizure; n = 32) were compared with age-matched peers (n = 30) with no history of such events using magnetic resonance imaging. Glycemic control (evaluated based on glycated hemoglobin [HbA(lc)] level), episodes of diabetic ketoacidosis (DKA), and clinical variables were monitored continuously since diagnosis in all subjects. RESULTS: Mean HbA(lc) from diagnosis and the duration of T1DM were similar in those with and without a history of severe hypoglycemia (9.0% +/- 0.9% vs 8.8% +/- 0.9%; 7.2 +/- 2.7 years vs 6.7 +/- 2.3 years). A high prevalence of central nervous system (CNS) structural abnormalities was detected (29%), and mesial temporal sclerosis (MTS) was detected in 16% of the total sample (n = 62). The presence of MTS was not associated with a history of severe hypoglycemia or DKA. Analysis of brain matter volumes suggested relatively less gray matter density in those subjects with a history of severe hypoglycemia. CONCLUSIONS: Early age of onset of T1DM per se is associated with a high incidence of CNS abnormalities, particularly MTS, suggesting hippocampal damage. Early-onset severe hypoglycemia may have an effect on gray matter volume.