We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.

SUMMARY Although rod and cone photoreceptor cells in the vertebrate retina are anatomically connected or coupled by gap junctions, rod-cone coupling is thought to be weak. By using a combination of tracer labeling and electrical recording in the goldfish retina and tracer labeling in the mouse retina, we show that the retinal circadian clock, a local endogenous process, and not the retinal response to the visual environment, controls the extent and strength of rod-cone coupling by activating dopamine D2-like receptors in the day, so that rod-cone coupling is weak during the day but remarkably robust at night. The results demonstrate that circadian control of rod-cone coupling serves as a synaptic switch between the rod and cone pathways, so that cones and neurons postsynaptic to cones receive very dim light signals from rods at night, but not in the day. The increase in the strength and extent of rod-cone coupling at night may enhance the reliability of the rod light response and facilitate the detection of large dim objects.

Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wlds) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein.

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activityinduced neurodegeneration and injury-induced axonal degeneration 1,2 . Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heatshock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions. Injury-induced axonal degeneration is dramatically delayed in wallerian degeneration slow (Wld S ) mice, a mutant strain that over-expresses a chimaeric protein containing the NAD synthase NMNAT 3,4 . The Wld S chimaeric protein offers neuroprotection against axonal degeneration 2,5-7 as well as a variety of neurodegenerative conditions [8][9][10][11] . Wld S protein contains the amino (N)-terminal 70-amino-acid fragment of ubiquitination factor E4B ©2008 Nature Publishing GroupCorrespondence and requests for materials should be addressed to R.G. 4,14,15 . Drosophila contains only one NMNAT gene, whose overexpression delays axonal degeneration 2 . This study and our finding that NMNAT functions as a maintenance factor to protect against activity-induced neurodegeneration 1 suggest that NMNAT alone can protect against multiple neurodegenerative insults. Our recent finding that enzymatically inactive NMNAT retains neuroprotective capabilities also exposed a hitherto unknown molecular function 1 .To test if NMNAT is a general factor required for neuronal maintenance and protection, we first examined the effects of NMNAT overexpression in a Drosophila model for SCA1. Overexpression of wild-type NMNAT or enzyme-inactive NMNAT (NMNAT-WR) 1 suppresses the degenerative phenotypes induced by overexpression of Drosophila ataxin-1 (dAtx-1). It also offers moderate protection against the severe phenotypes caused by overexpression of human ataxin-1 with an expanded (82) poly-glutamine tract (hAtx-1[82Q]) 16 (Fig. 1). These findings, and the observations that NMNAT protects from axonal injury 2 , from photoreceptor injury caused by intense light, and that its loss c...

Schizophrenia is a severe mental disorder that affects 0.5–1% of the population worldwide. Current diagnostic methods are based on psychiatric interviews, which are subjective in nature. The lack of disease biomarkers to support objective laboratory tests has been a long-standing bottleneck in the clinical diagnosis and evaluation of schizophrenia. Here we report a global metabolic profiling study involving 112 schizophrenic patients and 110 healthy subjects, who were divided into a training set and a test set, designed to identify metabolite markers. A panel of serum markers consisting of glycerate, eicosenoic acid, β-hydroxybutyrate, pyruvate and cystine was identified as an effective diagnostic tool, achieving an area under the receiver operating characteristic curve (AUC) of 0.945 in the training samples (62 patients and 62 controls) and 0.895 in the test samples (50 patients and 48 controls). Furthermore, a composite panel by the addition of urine β-hydroxybutyrate to the serum panel achieved a more satisfactory accuracy, which reached an AUC of 1 in both the training set and the test set. Multiple fatty acids and ketone bodies were found significantly (P<0.01) elevated in both the serum and urine of patients, suggesting an upregulated fatty acid catabolism, presumably resulting from an insufficiency of glucose supply in the brains of schizophrenia patients.

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. We have isolated an exocyst component, sec15, in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. Our data suggest a role of Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions.

As an adaptation to infrequent access to water, terrestrial mammals produce urine that is hyperosmotic to plasma. To prevent osmotic diuresis by the large quantity of urea generated by protein catabolism, the kidney epithelia contain facilitative urea transporters (UTs) that allow rapid equilibration between the urinary space and the hyperosmotic interstitium. Here we report the first X-ray crystal structure of a mammalian UT, UT-B, at a resolution of 2.36 Å. UT-B is a homotrimer and each protomer contains a urea conduction pore with a narrow selectivity filter. Structural analyses and molecular dynamics simulations showed that the selectivity filter has two urea binding sites separated by an approximately 5.0 kcal∕mol energy barrier. Functional studies showed that the rate of urea conduction in UT-B is increased by hypoosmotic stress, and that the site of osmoregulation coincides with the location of the energy barrier. channels | membrane proteins | renal physiology | osmosensing