Rei Morikawa

Identification of a Novel Member of the Carboxylesterase Family That Hydrolyzes Triacylglycerol: A Potential Role in Adipocyte Lipolysis

Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose tissue, are not fully understood. A database search for enzymes with α/β hydrolase folds, the GXSXG motif for serine esterase and the His-Gly dipeptide motif, has provided a previously unannotated gene that is induced during 3T3-L1 adipocytic differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4% identity, we have tentatively designated this enzyme as TGH-2 and the original TGH as TGH-1. TGH-2 is also similar to TGH-1 in terms of tissue distribution, subcellular localization, substrate specificity, and regulation. Both enzymes are predominantly expressed in liver, adipose tissue, and kidney. In adipocytes, they are localized in microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein, cholesteryl oleate, or phospholipids; hydrolysis of short-chain fatty acid ester was 30,000-fold more efficient than that of long-chain fatty acid triacylglycerol. Fasting increased the expression of both genes in white adipose tissue, whereas refeeding suppressed their expression. RNA silencing of TGH-2 reduced isoproterenol-stimulated glycerol release by 10% in 3T3-L1 adipocytes, while its overexpression increased the glycerol release by 20%. Thus, TGH-2 may make a contribution to adipocyte lipolysis during period of increased energy demand.

The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway

To cover the receptive field completely and non‐redundantly, neurons of certain functional groups arrange tiling of their dendrites. In Drosophila class IV dendrite arborization (da) neurons, the NDR family kinase Tricornered (Trc) is required for homotypic repulsion of dendrites that facilitates dendritic tiling. We here report that Sin1, Rictor, and target of rapamycin (TOR), components of the TOR complex 2 (TORC2), are required for dendritic tiling of class IV da neurons. Similar to trc mutants, dendrites of sin1 and rictor mutants show inappropriate overlap of the dendritic fields. TORC2 components physically and genetically interact with Trc, consistent with a shared role in regulating dendritic tiling. Moreover, TORC2 is essential for Trc phosphorylation on a residue that is critical for Trc activity in vivo and in vitro. Remarkably, neuronal expression of a dominant active form of Trc rescues the tiling defects in sin1 and rictor mutants. These findings suggest that TORC2 likely acts together with the Trc signalling pathway to regulate the dendritic tiling of class IV da neurons, and thus uncover the first neuronal function of TORC2 in vivo.

Compartmentalized Calcium Transients Trigger Dendrite Pruning in Drosophila Sensory Neurons

Dendritic Pruning During metamorphosis, Drosophila sensory neurons eliminate their dendritic trees, but axons and soma remain intact. Kanamori et al. (p. 1475, published online 30 May) demonstrate that compartmentalized calcium transients in dendrites function as the spatiotemporal cue for pruning of unwanted branches. Such a localized calcium signal, induced by a local elevation of branch excitability, activates calcium-dependent proteinases and eventually causes branch death. During fruit fly metamorphosis, dendritic calcium signaling defines the branches to be eliminated in sensory neurons. Dendrite pruning is critical for sculpting the final connectivity of neural circuits as it removes inappropriate projections, yet how neurons can selectively eliminate unnecessary dendritic branches remains elusive. Here, we show that calcium transients that are compartmentalized in specific dendritic branches act as temporal and spatial cues to trigger pruning in Drosophila sensory neurons. Calcium transients occurred in local dendrites at ~3 hours before branch elimination. In dendritic branches, intrinsic excitability increased locally to activate calcium influx via the voltage-gated calcium channels (VGCCs), and blockade of the VGCC activities impaired pruning. Further genetic analyses suggest that the calcium-activated protease calpain functions downstream of the calcium transients. Our findings reveal the importance of the compartmentalized subdendritic calcium signaling in spatiotemporally selective elimination of dendritic branches.

Dendrite reshaping of adult Drosophila sensory neurons requires matrix metalloproteinase-mediated modification of the basement membranes.

In response to changes in the environment, dendrites from certain neurons change their shape, yet the mechanism remains largely unknown. Here we show that dendritic arbors of adult Drosophila sensory neurons are rapidly reshaped from a radial shape to a lattice-like shape within 24 hr after eclosion. This radial-to-lattice reshaping arises from rearrangement of the existing radial branches into the lattice-like pattern, rather than extensive dendrite pruning followed by regrowth of the lattice-shaped arbors over the period. We also find that the dendrite reshaping is completely blocked in mutants for the matrix metalloproteinase (Mmp) 2. Further genetic analysis indicates that Mmp2 promotes the dendrite reshaping through local degradation of the basement membrane upon which dendrites of the sensory neurons innervate. These findings suggest that regulated proteolytic alteration of the extracellular matrix microenvironment might be a fundamental mechanism to drive a large-scale change of dendritic structures during reorganization of neuronal circuits.

Intracellular Phospholipase A1γ (iPLA1γ) Is a Novel Factor Involved in Coat Protein Complex I- and Rab6-independent Retrograde Transport between the Endoplasmic Reticulum and the Golgi Complex

The mammalian intracellular phospholipase A1 (iPLA1) family consists of three members, iPLA1α/PA-PLA1, iPLA1β/p125, and iPLA1γ/KIAA0725p. Although iPLA1β has been implicated in organization of the ER-Golgi compartments, little is known about the physiological role of its closest paralog, iPLA1γ. Here we show that iPLA1γ mediates a specific retrograde membrane transport pathway between the endoplasmic reticulum (ER) and the Golgi complex. iPLA1γ appeared to be localized to the cytosol, the cis-Golgi, and the ER-Golgi intermediate compartment (ERGIC). Time-lapse microscopy revealed that a population of GFP-iPLA1γ was associated with transport carriers moving out from the Golgi complex. Knockdown of iPLA1γ expression by RNAi did not affect the anterograde transport of VSVGts045 but dramatically delayed two types of Golgi-to-ER retrograde membrane transport; that is, transfer of the Golgi membrane into the ER in the presence of brefeldin A and delivery of cholera toxin B subunit from the Golgi complex to the ER. Notably, knockdown of iPLA1γ did not impair COPI- and Rab6-dependent retrograde transports represented by ERGIC-53 recycling and ER delivery of Shiga toxin, respectively. Thus, iPLA1γ is a novel membrane transport factor that contributes to a specific Golgi-to-ER retrograde pathway distinct from presently characterized COPI- and Rab6-dependent pathways.

Different levels of the Tripartite motif protein, Anomalies in sensory axon patterning (Asap), regulate distinct axonal projections of Drosophila sensory neurons

The axonal projection pattern of sensory neurons typically is regulated by environmental signals, but how different sensory afferents can establish distinct projections in the same environment remains largely unknown. Drosophila class IV dendrite arborization (C4da) sensory neurons project subtype-specific axonal branches in the ventral nerve cord, and we show that the Tripartite motif protein, Anomalies in sensory axon patterning (Asap) is a critical determinant of the axonal projection patterns of different C4da neurons. Asap is highly expressed in C4da neurons with both ipsilateral and contralateral axonal projections, but the Asap level is low in neurons that have only ipsilateral projections. Mutations in asap cause a specific loss of contralateral projections, whereas overexpression of Asap induces ectopic contralateral projections in C4da neurons. We also show by biochemical and genetic analysis that Asap regulates Netrin signaling, at least in part by linking the Netrin receptor Frazzled to the downstream effector Pico. In the absence of Asap, the sensory afferent connectivity within the ventral nerve cord is disrupted, resulting in specific larval behavioral deficits. These results indicate that different levels of Asap determine distinct patterns of axonal projections of C4da neurons by modulating Netrin signaling and that the Asap-mediated axonal projection is critical for assembly of a functional sensory circuit.

Trim9 Regulates Activity-Dependent Fine-Scale Topography in Drosophila

Topographic projection of afferent terminals into 2D maps in the CNS is a general strategy used by the nervous system to encode the locations of sensory stimuli. In vertebrates, it is known that although guidance cues are critical for establishing a coarse topographic map, neural activity directs fine-scale topography between adjacent afferent terminals [1-4]. However, the molecular mechanism underlying activity-dependent regulation of fine-scale topography is poorly understood. Molecular analysis of the spatial relationship between adjacent afferent terminals requires reliable localization of the presynaptic terminals of single neurons as well as genetic manipulations with single-cell resolution in vivo. Although both requirements can potentially be met in Drosophila melanogaster [5, 6], no activity-dependent topographic system has been identified in flies [7]. Here we report a topographic system that is shaped by neuronal activity in Drosophila. With this system, we found that topographic separation of the presynaptic terminals of adjacent nociceptive neurons requires different levels of Trim9, an evolutionarily conserved signaling molecule [8-11]. Neural activity regulates Trim9 protein levels to direct fine-scale topography of sensory afferents. This study offers both a novel mechanism by which neural activity directs fine-scale topography of axon terminals and a new system to study this process at single-neuron resolution.

Phospholipase A1 Assays Using a Radiolabeled Substrate and Mass Spectrometry

Although a number of phospholipase A1s (PLA1s) have been identified in the recent decade, the physiological functions of PLA1s remain almost elusive. The major reason for this is the poor availability of assay methods. In many studies, radiolabeled phospholipid substrates have been used to measure PLA1 activity. This chapter describes the conventional PLA1 assay using a radiolabeled substrate and a novel PLA1 assay using electrospray ionization mass spectrometry.

Common variable immune deficiency in a Pomeranian with Pneumocystis carinii pneumonia

A Pomeranian dog, 1 year- and 8 month-old neutered female, was presented with persistent respiratory distress and recurrent generalized demodicosis. Physical examination revealed cyanosis, rough respiratory sounds, multifocal alopecia and dermal erosions on the dorsal side of the forelimbs, perineal area and skin around the eyes. A severe diffuse interstitial lung pattern was observed on thoracic radiographs. The blood examination revealed neutrophilia and hypoglobulinemia. Serum immunoglobulin concentrations of IgG and IgA were low. Histopathological examination revealed severe diffuse interstitial pneumonia with Pneumocystis carinii infection. Severe lymphoid depletion was observed in the spleen and other organs with lymphoid follicles consisted mainly of CD3-positive T cells and few cells of B-cell lineage. B-cell hypoplasia with subsequent antibody deficiency was suspected.