Motor deficit in a Drosophila model of mucolipidosis type IV due to defective clearance of apoptotic cells.

Disruption of the Transient Receptor Potential (TRP) mucolipin 1 (TRPML1) channel results in the neurodegenerative disorder mucolipidosis type IV (MLIV), a lysosomal storage disease with severe motor impairments. The mechanisms underlying MLIV are poorly understood and there is no treatment. Here, we report a Drosophila MLIV model, which recapitulates the key disease features, including abnormal intracellular accumulation of macromolecules, motor defects, and neurodegeneration. The basis for the buildup of macromolecules was defective autophagy, which resulted in oxidative stress and impaired synaptic transmission. Late-apoptotic cells accumulated in trpml mutant brains, suggesting diminished cell clearance. The accumulation of late-apoptotic cells and motor deficits were suppressed by expression of trpml(+) in neurons, glia, or hematopoietic cells. We conclude that the neurodegeneration and motor defects result primarily from decreased clearance of apoptotic cells. Since hematopoietic cells in humans are involved in clearance of apoptotic cells, our results raise the possibility that bone marrow transplantation may limit the progression of MLIV.

Light-induced translocation of Drosophila visual Arrestin2 depends on Rac2.

Photoreceptor cells are remarkable in their ability to adjust their sensitivity to light over a wide range of intensities. Rapid termination of the photoresponse is achieved in part by shuttling proteins in and out of the light-transducing compartment of the photoreceptor cells. One protein that undergoes light-dependent translocation is the rhodopsin regulatory protein arrestin. However, the mechanisms coupling rhodopsin to arrestin movement are poorly understood. Here we show that light-dependent shuttling of the major arrestin in Drosophila photoreceptor cells, Arrestin2 (Arr2), occurs independently of known elements of the phototransduction cascade. Disruptions of the trimeric G protein, phospholipase Cbeta, the TRP channel, or the Na(+)/Ca(2+) exchanger did not influence Arr2 localization. Rather, we found that loss of the small GTPase Rac2 severely impaired Arr2 movement and prolonged the termination of the photoresponse. Our findings demonstrate that light-induced translocation of Arr2 occurs through a noncanonical rhodopsin/Rac2 pathway, which is distinct from the classical phototransduction cascade.

Characterization and turnover of CD73/IP(3)R3-positive microvillar cells in the adult mouse olfactory epithelium.

The main olfactory epithelium consists of 4 major cell types: sensory neurons, supporting cells, microvillar cells, and basal progenitor cells. Several populations of microvillar olfactory cells have been described, whose properties are not yet fully understood. In this study, we aimed to clarify the classification of microvillar cells by introducing a specific marker, CD73. Furthermore, we investigated the turnover of CD73-microvillar cells during adult life. Using direct and indirect immunofluorescence in adult main olfactory epithelium, we first demonstrate that ecto-5'-nucleotidase (CD73) is a reliable marker for microvillar cells reported previously to express phospholipase C ß2 (PLC ß2) along with type 3 IP(3) receptors (IP(3)R3) and transient receptor potential channels 6 (TRPC6), as well as for cells labeled by transgenic expression of tauGFP driven by the IP(3)R3 promoter. The ubiquitous CD73 immunoreactivity in the microvilli of these 2 cell populations indicates that they correspond to the same cell type (CD73-microvillar cell), endowed with a signal transduction cascade mobilizing Ca(++) from intracellular stores. These microvillar cells respond to odors, possess a basal process, and do not degenerate after bulbectomy, suggesting that they contribute to cellular homeostasis in the olfactory epithelium. Next, we examined whether CD73-microvillar cells undergo turnover in the adult olfactory epithelium. By combining CD73 immunofluorescence and BrdU pulse labeling, we show delayed BrdU incorporation in a small fraction of CD73-positive microvillar cells, which persists for several weeks after BrdU administration. These findings indicate that CD73-microvillar cells likely differentiate from proliferating progenitor cells and have a slow turnover despite their apical position in the olfactory epithelium. These combined properties are unique among olfactory cells, in line with the possibility that they might regulate cellular homeostasis driven by extracellular ATP and adenosine.

Dependence on a retinophilin/myosin complex for stability of PKC and INAD and termination of phototransduction.

Normal termination of signaling is essential to reset signaling cascades, especially those such as phototransduction that are turned on and off with great rapidity. Genetic approaches in Drosophila led to the identification of several proteins required for termination, including protein kinase C (PKC), NINAC (neither inactivation nor afterpotential C) p174, which consists of fused protein kinase and myosin domains, and a PDZ (postsynaptic density-95/Discs Large/zona occludens-1) scaffold protein, INAD (inactivation no afterpotential D). Here, we describe a mutation affecting a poorly characterized but evolutionarily conserved protein, Retinophilin (Retin), which is expressed primarily in the phototransducing compartment of photoreceptor cells, the rhabdomeres. Retin and NINAC formed a complex and were mutually dependent on each other for expression. Loss of retin resulted in an age-dependent impairment in termination of phototransduction. Mutations that affect termination of the photoresponse typically lead to a reduction in levels of the major rhodopsin (Rh1) to attenuate signaling. Consistent with the slower termination in retin(1), the mutant photoreceptor cells exhibited increased endocytosis of Rh1 and a decline in Rh1 protein. The slower termination in retin(1) was a consequence of a cascade of defects, which began with the reduction in NINAC p174 levels. The diminished p174 concentration caused a decrease in INAD. Because PKC requires interaction with INAD for protein stability, this leads to reduction in PKC levels. The decline in PKC was age dependent and paralleled the onset of the termination phenotype in retin(1) mutant flies. We conclude that the slower termination of the photoresponse in retin(1) resulted from a requirement for the Retin/NINAC complex for stability of INAD and PKC.

High-dose folic acid pretreatment blunts cardiac dysfunction during ischemia coupled to maintenance of high-energy phosphates and reduces postreperfusion injury.

BACKGROUND: The B vitamin folic acid (FA) is important to mitochondrial protein and nucleic acid synthesis, is an antioxidant, and enhances nitric oxide synthase activity. Here, we tested whether FA reduces myocardial ischemic dysfunction and postreperfusion injury. METHODS AND RESULTS: Wistar rats were pretreated with either FA (10 mg/d) or placebo for 1 week and then underwent in vivo transient left coronary artery occlusion for 30 minutes with or without 90 minutes of reperfusion (total n=131; subgroups used for various analyses). FA (4.5x10(-6) mol/L i.c.) pretreatment and global ischemia/reperfusion (30 minutes/30 minutes) also were performed in vitro (n=28). After 30 minutes of ischemia, global function declined more in controls than in FA-pretreated rats (Delta dP/dtmax, -878+/-586 versus -1956+/-351 mm Hg/s placebo; P=0.03), and regional thickening was better preserved (37.3+/-5.3% versus 5.1+/-0.6% placebo; P=0.004). Anterior wall perfusion fell similarly (-78.4+/-9.3% versus -71.2+/-13.8% placebo at 30 minutes), yet myocardial high-energy phosphates ATP and ADP reduced by ischemia in controls were better preserved by FA pretreatment (ATP: control, 2740+/-58 nmol/g; ischemia, 947+/-55 nmol/g; ischemia plus FA, 1332+/-101 nmol/g; P=0.02). Basal oxypurines (xanthine, hypoxanthine, and urate) rose with FA pretreatment but increased less during ischemia than in controls. Ischemic superoxide generation declined (3124+/-280 cpm/mg FA versus 5898+/-474 cpm/mg placebo; P=0.001). After reperfusion, FA-treated hearts had smaller infarcts (3.8+/-1.2% versus 60.3+/-4.1% placebo area at risk; P<0.002) and less contraction band necrosis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positivity, superoxide, and nitric oxide synthase uncoupling. Infarct size declined similarly with 1 mg/d FA. CONCLUSIONS: FA pretreatment blunts myocardial dysfunction during ischemia and ameliorates postreperfusion injury. This is coupled to preservation of high-energy phosphates, reducing subsequent reactive oxygen species generation, eNOS-uncoupling, and postreperfusion cell death.

The sense of smell, its signalling pathways, and the dichotomy of cilia and microvilli in olfactory sensory cells.

Smell is often regarded as an ancillary perception in primates, who seem so dominated by their sense of vision. In this paper, we will portray some aspects of the significance of olfaction to human life and speculate on what evolutionary factors contribute to keeping it alive. We then outline the functional architecture of olfactory sensory neurons and their signal transduction pathways, which are the primary detectors that render olfactory perception possible. Throughout the phylogenetic tree, olfactory neurons, at their apical tip, are either decorated with cilia or with microvilli. The significance of this dichotomy is unknown. It is generally assumed that mammalian olfactory neurons are of the ciliary type only. The existence of so-called olfactory microvillar cells in mammals, however, is well documented, but their nature remains unclear and their function orphaned. This paper discusses the possibility, that in the main olfactory epithelium of mammals ciliated and microvillar sensory cells exist concurrently. We review evidence related to this hypothesis and ask, what function olfactory microvillar cells might have and what signalling mechanisms they use.

Novel role of cystic fibrosis transmembrane conductance regulator in maintaining adult mouse olfactory neuronal homeostasis.

The olfactory epithelium (OE) of mice deficient in cystic fibrosis transmembrane conductance regulator (CFTR) exhibits ion transport deficiencies reported in human CF airways, as well as progressive neuronal loss, suggesting defects in olfactory neuron homeostasis. Microvillar cells, a specialized OE cell-subtype, have been implicated in maintaining tissue homeostasis. These cells are endowed with a PLCß2/IP3 R3/TRPC6 signal transduction pathway modulating release of neuropeptide Y (NPY), which stimulates OE stem cell activity. It is unknown, however, whether microvillar cells also mediate the deficits observed in CFTR-null mice. Here we show that Cftr mRNA in mouse OE is exclusively localized in microvillar cells and CFTR immunofluorescence is coassociated with the scaffolding protein NHERF-1 and PLCß2 in microvilli. In CFTR-null mice, PLCß2 was undetectable, NHERF-1 mislocalized, and IP3 R3 more intensely stained, along with increased levels of NPY, suggesting profound alteration of the PLCß2/IP3 R3 signaling pathway. In addition, basal olfactory neuron homeostasis was altered, shown by increased progenitor cell proliferation, differentiation, and apoptosis and by reduced regenerative capacity following methimazole-induced neurodegeneration. The importance of CFTR in microvillar cells was further underscored by decreased thickness of the OE mucus layer and increased numbers of immune cells within this tissue in CFTR-KO mice. Finally, we observed enhanced immune responses to an acute viral-like infection, as well as hyper-responsiveness to chemical and physical stimuli applied intranasally. Taken together, these data strengthen the notion that microvillar cells in the OE play a key role in maintaining tissue homeostasis and identify several mechanisms underlying this regulation through the multiple functions of CFTR.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells.

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2(-/-) mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2(-/-) were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2(-/-) mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light.

Neuropeptide Y in the olfactory microvillar cells.

This paper examines a possible role of microvillar cells in coordinating cell death and regeneration of olfactory epithelial neurons. The olfactory neuroepithelium of mammals is a highly dynamic organ. Olfactory neurons periodically degenerate by apoptosis and as a consequence of chemical or physical damage. To compensate for this loss of cells, the olfactory epithelium maintains a lifelong ability to regenerate from a pool of resident multipotent stem cells. To assure functional continuity and histological integrity of the olfactory epithelium over a period of many decades, apoptosis and regeneration require to be precisely coordinated. Among the factors that have been implicated in mediating this regulation is the neuropeptide Y (NPY). Knockout mice that lack functional expression of this neurogenic peptide show defects in embryonic development of the olfactory epithelium and in its ability to regenerate in the adult. Here we show that, in postnatal olfactory epithelia, NPY is exclusively expressed by a specific population of microvillar cells. We previously characterized these cells as a novel type of putative chemosensory cells, which are provided with a phosphatidyl-inositol-mediated signal transduction cascade. Our findings allow for the first time to suggest that microvillar cells are involved in connecting apoptosis to neuronal regeneration by stimulus-induced release of NPY.

Morituri te salutant? Olfactory signal transduction and the role of phosphoinositides.

During the past 150 years, researchers have investigated the cellular, physiological, and molecular mechanisms underlying the sense of smell. Based on these efforts, a conclusive model of olfactory signal transduction in the vertebrate's nose is now available, spanning from G-protein-mediated odorant receptors to ion channels, which are linked by a cyclic adenosine 3',5'-monophosphate-mediated signal transduction cascade. Here we review some historical milestones in the chronology of olfactory research, particularly emphasising the role of cyclic nucleotides and inositol trisphosphate as alternative second messengers in olfactory cells. We will describe the functional anatomy of the nose, outline the cellular composition of the olfactory epithelium, and describe the discovery of the molecular backbone of the olfactory signal transduction cascade. We then summarize our current model, in which cyclic adenosine monophosphate is the sole excitatory second messenger in olfactory sensory neurons. Finally, a possible significance of microvillous olfactory epithelial cells and inositol trisphosphate in olfaction will be discussed.

Phosphatidyl-inositide signalling proteins in a novel class of sensory cells in the mammalian olfactory epithelium.

Ciliated sensory neurons, supporting cells and basal stem cells represent major cellular components of the main olfactory epithelium in mammals. Here we describe a novel class of sensory cells in the olfactory neuroepithelium. The cells express phospholipase C beta-2 (PLC beta2), transient receptor potential channels 6 (TRPC6) and inositol 3, 4, 5-trisphosphate receptors type III (InsP3R-III). Unlike ciliated olfactory neurons, they express neither olfactory marker protein nor centrin, adenylyl cyclase or cyclic nucleotide-gated cation channels. Typical components of the cytoskeleton of microvilli, ezrin and actin are found co-localized with PLC beta2 and TRPC6 in apical protrusions of the cells. In Ca2+-imaging experiments, the cells responded to odours. They express neuronal marker proteins and possess an axon-like process, but following bulbectomy the cells do not degenerate. Our results suggest a novel class of microvillous secondary chemosensory cells in the mammalian olfactory system. These cells, which utilize phosphatidyl-inositides in signal transduction, represent about 5% of all olfactory cells. Their abundance indicates that they play an important role in stimulus-dependent functions and/or the regeneration of the olfactory system.