RESUMEN
Glia derived secretory factors play diverse roles in supporting the development, physiology, and stress responses of the central nervous system (CNS). Through transcriptomics and imaging analyses, we have identified Obp44a as one of the most abundantly produced secretory proteins from Drosophila CNS glia. Protein structure homology modeling and Nuclear Magnetic Resonance (NMR) experiments reveal Obp44a as a fatty acid binding protein (FABP) with a high affinity towards long-chain fatty acids in both native and oxidized forms. Further analyses demonstrate that Obp44a effectively infiltrates the neuropil, traffics between neuron and glia, and is secreted into hemolymph, acting as a lipid chaperone and scavenger to regulate lipid and redox homeostasis in the developing brain. In agreement with this essential role, deficiency of Obp44a leads to anatomical and behavioral deficits in adult animals and elevated oxidized lipid levels. Collectively, our findings unveil the crucial involvement of a noncanonical lipid chaperone to shuttle fatty acids within and outside the brain, as needed to maintain a healthy brain lipid environment. These findings could inspire the design of novel approaches to restore lipid homeostasis that is dysregulated in CNS diseases.
RESUMEN
The construction and maturation of the postsynaptic apparatus are crucial for synapse and dendrite development. The fundamental mechanisms underlying these processes are most often studied in glutamatergic central synapses in vertebrates. Whether the same principles apply to excitatory cholinergic synapses, such as those found in the insect central nervous system, is not known. To address this question, we investigated a group of projection neurons in the Drosophila larval visual system, the ventral lateral neurons (LNvs), and identified nAchRα1 (Dα1) and nAchRα6 (Dα6) as the main functional nicotinic acetylcholine receptor (nAchR) subunits in the larval LNvs. Using morphological analyses and calcium imaging studies, we demonstrated critical roles of these two subunits in supporting dendrite morphogenesis and synaptic transmission. Furthermore, our RNA sequencing analyses and endogenous tagging approach identified distinct transcriptional controls over the two subunits in the LNvs, which led to the up-regulation of Dα1 and down-regulation of Dα6 during larval development as well as to an activity-dependent suppression of Dα1 Additional functional analyses of synapse formation and dendrite dynamics further revealed a close association between the temporal regulation of individual nAchR subunits and their sequential requirements during the cholinergic synapse maturation. Together, our findings support transcriptional control of nAchR subunits as a core element of developmental and activity-dependent regulation of central cholinergic synapses.
Asunto(s)
Neuronas Colinérgicas/metabolismo , Dendritas/metabolismo , Proteínas de Drosophila/biosíntesis , Morfogénesis , Receptores Nicotínicos/biosíntesis , Sinapsis/metabolismo , Transmisión Sináptica , Animales , Drosophila melanogaster , Larva/metabolismoRESUMEN
Lipid shuttling between neurons and glia contributes to the development, function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, we perform combined genetic, transcriptome, and biochemical analyses in the developing Drosophila larval brain. Here we report, the astrocyte-derived secreted lipocalin Glial Lazarillo (GLaz), a homolog of human Apolipoprotein D (APOD), and its neuronal receptor, the brain-specific short isoforms of Drosophila lipophorin receptor 1 (LpR1-short), cooperatively mediate neuron-glia lipid shuttling and support dendrite morphogenesis. The isoform specificity of LpR1 defines its distribution, binding partners, and ability to support proper dendrite growth and synaptic connectivity. By demonstrating physical and functional interactions between GLaz/APOD and LpR1, we elucidate molecular pathways mediating lipid trafficking in the fly brain, and provide in vivo evidence indicating isoform-specific expression of lipoprotein receptors as a key mechanism for regulating cell-type specific lipid recruitment.
Asunto(s)
Apolipoproteínas/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Animales Modificados Genéticamente , Apolipoproteínas/genética , Transporte Biológico , Encéfalo/citología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Perfilación de la Expresión Génica , Humanos , Larva/genética , Larva/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in this simple circuit, we focus on investigating the physiological properties and behavioral relevance of larval visual interneurons. We find that the ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Gαo signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, our studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system.
Asunto(s)
Drosophila melanogaster/fisiología , Receptores Muscarínicos/metabolismo , Transducción de Señal , Vías Visuales/metabolismo , Animales , Conducta Animal/efectos de la radiación , Calcio/metabolismo , Drosophila melanogaster/efectos de la radiación , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ácido Glutámico/metabolismo , Interneuronas/metabolismo , Interneuronas/efectos de la radiación , Larva/efectos de la radiación , Luz , Neurópilo/metabolismo , Neurópilo/efectos de la radiación , Terminales Presinápticos/metabolismo , Terminales Presinápticos/efectos de la radiaciónRESUMEN
Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKC epsilon (at the C-terminal hydrophobic motif) and PKC delta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKC delta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKC delta kinase activity is attenuated when PKC delta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC delta and PKC epsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC delta and WT-PKC epsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC delta is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKC epsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKC epsilon in nPKC activation loop phosphorylations and PKC delta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKC delta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.
Asunto(s)
Miocardio/citología , Proteína Quinasa C/química , Proteína Quinasa C/fisiología , Adenoviridae/genética , Sitio Alostérico , Secuencias de Aminoácidos , Animales , Células Cultivadas , Secuencia Conservada , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Técnicas de Transferencia de Gen , Genes Dominantes , Immunoblotting , Cinética , Modelos Biológicos , Fosforilación , Isoformas de Proteínas , Proteína Quinasa C/metabolismo , Proteína Quinasa C-delta , Proteína Quinasa C-epsilon , Transporte de Proteínas , Ratas , Ratas Wistar , Transducción de Señal , Especificidad por Sustrato , Factores de Tiempo , TransfecciónRESUMEN
Proteases elaborated by inflammatory cells in the heart would be expected to drive cardiac fibroblasts to proliferate, but protease-activated receptor (PAR) function in cardiac fibroblasts has never been considered. This study demonstrates that PAR-1 is the only known PAR family member functionally expressed by cardiac fibroblasts and that PAR-1 activation by thrombin leads to increased DNA synthesis in cardiac fibroblasts. The increase in DNA synthesis induced by PAR-1 substantially exceeds the effects of other G protein-coupled receptor agonists in this cell type. PAR-1 stimulates phosphoinositide hydrolysis and mobilizes intracellular calcium via pertussis toxin (PTX)-sensitive and PTX-insensitive pathways. Activation of PAR-1 leads to an increase in Src, Fyn, and epidermal growth factor receptor (EGFR) phosphorylation, with EGFR receptor transactivation by Src family kinases the major mechanism for PAR-1-dependent activation of extracellular signal-regulated kinase, p38-mitogen-activated protein kinase, and protein kinase B. Activation of PAR-1 also leads to an increase in DNA synthesis. PAR-1 signaling is highly contextual in nature, inasmuch as PAR-1 activates extracellular signal-regulated kinase and only weakly stimulates protein kinase B via a pathway that does not involve EGFR transactivation in cardiomyocytes. PAR-1 responses in cardiac fibroblasts and cardiomyocytes are predicted to contribute importantly to remodeling during cardiac injury and/or inflammation.