Drosophila Vision Depends on Carcinine Uptake by an Organic Cation Transporter.

Recycling of neurotransmitters is essential for sustained neuronal signaling, yet recycling pathways for various transmitters, including histamine, remain poorly understood. In the first visual ganglion (lamina) of Drosophila, photoreceptor-released histamine is taken up into perisynaptic glia, converted to carcinine, and delivered back to the photoreceptor for histamine regeneration. Here, we identify an organic cation transporter, CarT (carcinine transporter), that transports carcinine into photoreceptors during histamine recycling. CarT mediated in vitro uptake of carcinine. Deletion of the CarT gene caused an accumulation of carcinine in laminar glia accompanied by a reduction in histamine, resulting in abolished photoreceptor signal transmission and blindness in behavioral assays. These defects were rescued by expression of CarT cDNA in photoreceptors, and they were reproduced by photoreceptor-specific CarT knockdown. Our findings suggest a common role for the conserved family of CarT-like transporters in maintaining histamine homeostasis in both mammalian and fly brains.

The putative Na⁺/Cl⁻-dependent neurotransmitter/osmolyte transporter inebriated in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis.

Most organisms are able to maintain systemic water homeostasis over a wide range of external or dietary osmolarities. The excretory system, composed of the kidneys in mammals and the Malpighian tubules and hindgut in insects, can increase water conservation and absorption to maintain systemic water homeostasis, which enables organisms to tolerate external hypertonicity or desiccation. However, the mechanisms underlying the maintenance of systemic water homeostasis by the excretory system have not been fully characterized. In the present study, we found that the putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter inebriated (ine) is expressed in the basolateral membrane of anterior hindgut epithelial cells. This was confirmed by comparison with a known basolateral localized protein, the α subunit of Na(+)-K(+) ATPase (ATPα). Under external hypertonicity, loss of ine in the hindgut epithelium results in severe dehydration without damage to the hindgut epithelial cells, implicating a physiological failure of water conservation/absorption. We also found that hindgut expression of ine is required for water conservation under desiccating conditions. Importantly, specific expression of ine in the hindgut epithelium can completely restore disrupted systemic water homeostasis in ine mutants under both conditions. Therefore, ine in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis.

Loss of Na(+)/K(+)-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration.

The activity of Na(+)/K(+)-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na(+)/K(+)-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na(+)/K(+)-ATPase deficiency to in vivo neurodegeneration has been lacking. In this study, we use Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na(+)/K(+) ATPase. Loss of ATPα, an α subunit of Na(+)/K(+)-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K(+). Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na(+)/K(+)-ATPase ß subunit, partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na(+)/K(+)-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na(+)/K(+) ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na(+)/K(+) ATPase deficiency-mediated neurodegeneration.

The chaperone activity and toxicity of ambroxol on Gaucher cells and normal mice.

Gaucher disease (GD), caused by a defect of acid ß-glucosidase (ß-Glu), is one of the most common sphingolipidoses. Recently, ambroxol, an FDA-approved drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified as a chemical chaperone for GD. In the present study, we investigated the chaperone activity and toxicity of ambroxol on both cultured GD patient cells and normal mice. We found that ambroxol treatment significantly increased N370S, F213I, N188S/G193W and R120W mutant ß-Glu activities in GD fibroblasts with low cytotoxicity. Additionally, we measured the ß-Glu activity in the tissues of normal mice which received water containing increasing concentrations of ambroxol ad libitum for one week. No serious adverse effect was observed during this experiment. Ambroxol significantly increased the ß-Glu activity in the spleen, heart and cerebellum of the mice. This result showed its oral availability and wide distribution and chaperone activity in the tissues, including the brain, and its lack of acute toxicity. These characteristics of ambroxol would make it a potential therapeutic chaperone in the treatment of GD with neurological manifestations.

Inwardly rectifying potassium channels in Drosophila.

Inwardly rectifying potassium channels (Kir) are a special subset of potassium selective ion channels which pass potassium more easily into rather than out of the cell. These channels mediate a variety of cellular functions, including control of membrane resting potential, maintenance of potassium homeostasis and regulation of cellular metabolism. Given the existence of fifteen Kir genes in mammals, current genetic studies using mutant animals that lack a single channel may have missed many important physiological functions of these channels due to gene redundancy. This issue can be circumvented by using a simple model organism like Drosophila, whose genome encodes only 3 Kir proteins. The sophisticated genetic approaches of Drosophila may also provide powerful tools to identify additional regulation mechanisms of Kir channels. Here we provide an overview of the progress made in elucidating the function of Drosophila Kir channels. The knowledge of Drosophila Kir channels may lead us to uncover novel functions and regulation mechanisms of human Kir channels and help on pathological studies of related diseases.

A Fluorescent sp2-iminosugar with pharmacological chaperone activity for gaucher disease: synthesis and intracellular distribution studies.

Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid ß-glucosidase (ß-glucocerebrosidase; ß-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal ß-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the ß-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms.

Chemical chaperone therapy: luciferase assay for screening of ß-galactosidase mutations.

ß-Galactosidosis is a group of disorder based on heterogeneous mutations of GLB1 gene coding for the lysosomal acid ß-galactosidase (ß-gal). A decrease of the ß-gal enzyme activity results in progressive accumulation of substrates in somatic cells, particularly in neurons, leading to severe neuronal dysfunction. We have previously reported that N-octyl-4-epi-ß-valienamine (NOEV), a chemical chaperone compound, stabilized various mutant human ß-gal proteins and increased residual enzyme activities in cultured fibroblasts from human patients. These data proved a potential therapeutic benefit of chemical chaperone therapy for patients with missense ß-gal. This effect is mutation specific. In this study, we have established a sensitive luciferase-based assay for measuring chaperone effect on mutant human ß-gal. A dinoflagellate luciferase (Dluc) cDNA was introduced to the C-terminus of human ß-gal. When COS7 cells expressing the Dluc-tagged human R201C ß-gal was treated with NOEV, there happened a remarkable increase of the mutant ß-gal activity. In the presence of NH(4)Cl, luciferase level in the medium increased in parallel with the enzyme activity in cell lysates. We also found that proteasome inhibitors enhance chaperone effect of NOEV. These results demonstrate that the luciferase-based assay is a reliable and convenient method for screening and evaluation of chaperone effects on human ß-gal mutants, and that it will be a useful tool for finding novel chaperone compounds in the future study.

The effect of N-octyl-ß-valienamine on ß-glucosidase activity in tissues of normal mice.

Gaucher disease (GD), mainly caused by a defect of acid ß-glucosidase (ß-Glu), is the most common sphingolipidosis. We have previously shown that a carbohydrate mimic N-octyl-ß-valienamine (NOV), an inhibitor of ß-Glu, could increase the protein level and enzyme activity of various mutant ß-Glu in cultured GD fibroblasts, suggesting that NOV acted as a pharmacological chaperone to accelerate transport and maturation of this mutant enzymes. In the present study, the NOV effect was evaluated for ß-Glu activity, tissue distribution and adverse effects in normal mice. We measured the ß-Glu activity in tissues of normal mice which received water containing increasing concentrations of NOV ad libitum for 1 week. Fluid intake and body weight were measured periodically throughout the study. Measurement of tissue NOV concentration, blood chemistry and urinalysis were performed at the end of the study. The results showed that NOV had no impact on the body weight but fluid intake in the 10mM NOV group mice decreased and there was a moderate increase in blood urea nitrogen (BUN). No other adverse effect was observed during this experiment. Tissue NOV concentration increased in all tissues examined with increasing NOV doses. No inhibitory effect of NOV on ß-Glu was observed. Furthermore, NOV increased the ß-Glu activity in the liver, spleen, muscle and cerebellum of the mice significantly. This study on NOV showed its oral availability and wide tissue distribution, including the brain and its lack of acute toxicity. These characteristics of NOV would make it a potential therapeutic chaperone in the treatment of GD with neurological manifestations and selected mutations.

The pharmacological chaperone effect of N-octyl-beta-valienamine on human mutant acid beta-glucosidases.

Gaucher's disease (GD), mainly caused by a defect of acid beta-glucosidase (beta-Glu), is the most common form of sphingolipidosis. We have previously shown that the carbohydrate mimic and inhibitor of beta-Glu, N-octyl-beta-valienamine (NOV), could increase the protein level and enzyme activity of various mutant beta-Glus in cultured GD fibroblasts and in COS cells, suggesting that NOV acts as a pharmacological chaperone to accelerate transport and maturation of these mutant enzymes. In present study, we continued to investigate the chaperone characteristics of NOV. More importantly, chaperone activities of NOV were evaluated in COS cells transiently expressing ten new, recombinant beta-Glu mutants with mutations located in domain I, II and III. NOV was only effective on the T369M mutation, located in domain III. As we suggested in a previous study, domain III may be a prerequisite for pharmacological rescue of the mutant beta-Glu by NOV. These characteristics of NOV could provide potential therapeutic chaperone properties that would be useful in the treatment of GD with neurological manifestations due to gene mutations in beta-Glu.

Chaperone activity of bicyclic nojirimycin analogues for Gaucher mutations in comparison with N-(n-nonyl)deoxynojirimycin.

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal beta-glucosidase (acid beta-Glu, beta-glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N-(n-nonyl)-1-deoxynojirimycin (NN-DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as "chemical chaperones" that facilitate transport and maturation of acid beta-Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp2 iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal beta-Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5-N,6-O-(N'-octyliminomethylidene-NJ (NOI-NJ), the 6-thio and 6-amino-6-deoxy derivatives (6S-NOI-NJ and 6N-NOI-NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI-GNJ, 6S-NOI-GNJ and 6N-NOI-GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI-NJ, 6S-NOI-NJ, 6N-NOI-NJ, and 6S-NOI-GNJ) were further evaluated in GD fibroblasts with various acid beta-Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp2 iminosugar were generally stronger than those observed for NN-DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of beta-Glu mutations, especially for neuronopathic forms of Gaucher disease.

Brainstem neuropathology in a mouse model of Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a neurovisceral lipid storage disorder characterized by progressive and widespread neurodegeneration. Although some characteristic symptoms of NPC result from brainstem dysfunction, little information is available about which brainstem structures are affected. In this study, the brainstems of mutant BALB/c NPC1-/- mice with a retroposon insertion in the NPC1 gene were examined for neuropathological changes. In the midbrain, the integrated optic density (IOD) and cell count density of tyrosine-hydroxylase (TH) immunostained neurons were decreased in the substantia nigra. In the pons, TH immunoreactivity in the locus ceruleus (LC) neurons was decreased, while the IOD and the neuronal density of choline acetyltransferase (ChAT)-immunostained neurons in the pedunculopontine tegmental nucleus were preserved. The ChAT immunoreactivity of the hypoglossal nucleus (12N) neurons was not decreased, but Klüver-Barrera staining showed that neuronal density in the nucleus of the solitary tract (NTS) was decreased. Klüver-Barrera and neuronal nuclei (NeuN) staining showed a decrease in neuronal density in the ventral cochlear nucleus, but not in the dorsal cochlear nucleus. Gliosis was widely identified by GFAP staining in various brainstem structures, including the superior and inferior colliculi, the rostral interstitial nucleus of the medial longitudinal fasciculus, the oculomotor complex, the medial geniculate nucleus, the nucleus ambiguus, and the 12N. However, GFAP expression was not augmented in the LC, the cochlear nucleus, or the NTS. These neuropathological findings suggest a basis for the neurological syndromes observed in NPC, such as rigidity, oculomotor symptoms, cataplexy and sleep disturbance, dysphagia, and perceptive deafness.

Neuroprotective effects of iron chelator Desferal on dopaminergic neurons in the substantia nigra of rats with iron-overload.

The aim of the present study was to investigate whether the iron chelator Desferal prevents the degeneration of dopaminergic neurons in the substantia nigra (SN) induced by iron-overload in rats. Using fast cyclic voltammetry, tyrosine hydroxylase (TH) immunohistochemistry, Perls' iron staining, and high-performance liquid chromatography-electrochemical detection, we measured the degeneration of dopaminergic neurons and increased iron content in the SN of rats overloaded with iron dextran and assessed the effects of treatment with Desferal. The results showed that iron dextran overload increased the iron content in the SN, decreased dopamine release and content, and reduced the numbers of TH-immunoreactive neurons. Treatment with Desferal prevented the increased iron content in the SN. As a result, dopamine release and content remained at almost normal levels, while the numbers of TH-immunoreactive neurons remained at control values. This study suggests that the iron chelator Desferal is neuroprotective against iron-overload, so iron chelators that can cross the blood-brain barrier may have the potential to treat cases where abnormal iron accumulation in the brain is associated with the degenerative processes, as in Parkinson's disease.