Robert Kreber

Molecular analysis of the para locus, a sodium channel gene in Drosophila

Previous behavioral, electrophysiological, pharmacological, and genetic studies of mutations of the para locus in Drosophila melanogaster suggested that these mutations alter the structure or function of sodium channels. To identify the protein encoded by this gene and to elucidate the molecular basis of the mutant phenotypes, genomic DNA from the para locus was cloned. Mutational lesions in nine different para alleles were mapped and found to be distributed over a region of 45 kb. Analysis of cDNAs revealed that the para locus comprises a minimum of 26 exons distributed over more than 60 kb of genomic DNA. The nucleotide sequence of the complementary DNA predicts a protein whose structure and amino acid sequence are extremely similar to those of vertebrate sodium channels. The results support the conclusion that para encodes a functionally predominant class of sodium channels in Drosophila neurons. Furthermore, the para transcript appears to undergo alternative splicing to produce several distinct subtypes of this channel.

Reduced sleep in Drosophila Shaker mutants.

Most of us sleep 7–8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3–4 h of sleep—a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.

The maleless protein associates with the X chromosome to regulate dosage compensation in drosophila

The maleless (mle) gene is one of four known regulatory loci required for increased transcription (dosage compensation) of X-linked genes in D. melanogaster males. A predicted mle protein (MLE) contains seven short segments that define a superfamily of known and putative RNA and DNA helicases. MLE, while present in the nuclei of both male and female cells, differs in its association with polytene X chromosomes in the two sexes. MLE is associated with hundreds of discrete sites along the length of the X chromosome in males and not in females. The predominant localization of MLE to the X chromosome in males makes it a strong candidate to be a direct regulator of dosage compensation.

Temperature-Sensitive Paralytic Mutations Demonstrate that Synaptic Exocytosis Requires SNARE Complex Assembly and Disassembly

The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

Retromer deficiency observed in Alzheimer's disease causes hippocampal dysfunction, neurodegeneration, and Aβ accumulation

Although deficiencies in the retromer sorting pathway have been linked to late-onset Alzheimers disease, whether these deficiencies underlie the disease remains unknown. Here we characterized two genetically modified animal models to test separate but related questions about the effects that retromer deficiency has on the brain. First, testing for cognitive defects, we investigated retromer-deficient mice and found that they develop hippocampal-dependent memory and synaptic dysfunction, which was associated with elevations in endogenous Aβ peptide. Second, testing for neurodegeneration and amyloid deposits, we investigated retromer-deficient flies expressing human wild-type amyloid precursor protein (APP) and human β-site APP-cleaving enzyme (BACE) and found that they develop neuronal loss and human Aβ aggregates. By recapitulating features of the disease, these animal models suggest that retromer deficiency observed in late-onset Alzheimers disease can contribute to disease pathogenesis.

Drosophila CAPS Is an Essential Gene that Regulates Dense-Core Vesicle Release and Synaptic Vesicle Fusion

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.

napts, a Mutation affecting sodium channel activity in Drosophila, Is an allele of mle a regulator of X chromosome transcription

napts is a recessive mutation that affects the level of sodium channel activity and, at high temperature, causes paralysis associated with a loss of action potentials. We show, by genetic complementation tests, germline transformation, and analysis of mutations, that napts is a gain-of-function mutation of mle, a gene required for X chromosome dosage compensation and male viability. Molecular analyses of nap and mle mutations indicate that mle+, nap+, and napts activities are encoded by the same open reading frame and suggest that napts is due to a single amino acid substitution. Although napts is known to act via para+, an X-linked sodium channel structural gene, its effect is not due to a simple defect in para+ dosage compensation.

Neural dysfunction and neurodegeneration in Drosophila Na+/K+ ATPase alpha subunit mutants

The Na+/K+ ATPase asymmetrically distributes sodium and potassium ions across the plasma membrane to generate and maintain the membrane potential in many cell types. Although these pumps have been hypothesized to be involved in various human neurological disorders, including seizures and neurodegeneration, direct genetic evidence has been lacking. Here, we describe novel mutations in the Drosophila gene encoding the α (catalytic) subunit of the Na+/K+ ATPase that lead to behavioral abnormalities, reduced life span, and severe neuronal hyperexcitability. These phenotypes parallel the occurrence of extensive, age-dependent neurodegeneration. We have also discovered that the ATPalpha transcripts undergo alternative splicing that substantially increases the diversity of potential proteins. Our data show that maintenance of neuronal viability is dependent on normal sodium pump activity and establishDrosophila as a useful model for investigating the role of the pump in human neurodegenerative and seizure disorders.

wasted away, a Drosophila mutation in triosephosphate isomerase, causes paralysis, neurodegeneration, and early death.

To identify genes required for maintaining neuronal viability, we screened our collection of Drosophila temperature-sensitive paralytic mutants for those exhibiting shortened lifespan and neurodegeneration. Here, we describe the characterization of wasted away (wstd), a recessive, hypomorphic mutation that causes progressive motor impairment, vacuolar neuropathology, and severely reduced lifespan. We demonstrate that the affected gene encodes the glycolytic enzyme, triosephosphate isomerase (Tpi). Mutations causing Tpi deficiency in humans are also characterized by progressive neurological dysfunction, neurodegeneration, and early death. In Tpi-deficient flies and humans, a decrease in ATP levels did not appear to cause the observed phenotypes because ATP levels remained normal. We also found no genetic evidence that the mutant Drosophila Tpi was misfolded or involved in aberrant protein–protein associations. Instead, we favor the hypothesis that mutations in Tpi lead to an accumulation of methylglyoxal and the consequent enhanced production of advanced glycation end products, which are ultimately responsible for the death and dysfunction of Tpi-deficient neurons. Our results highlight an essential protective role of Tpi and support the idea that advanced glycation end products may also contribute to pathogenesis of other neurological disorders.

Effects on Eye Color Mediated by DNA Injection into Drosophila Embryos

SummaryDrosophila embryos of v; bw constitution were injected with purified v+; bw+ (allo-) and v; bw (iso-) DNA solutions. Flies with partial formation of brown pigment were recovered from both kinds of DNA injections, and several stocks were successfully established.Six allo-DNA induced stocks and their progeny have the following characteristics: (1) an initial period of unstable transmission of the “information” for the altered phenotype; (2) expression of the altered phenotype is intermediate; (3) the level of phenotypic expression varies from fly to fly within a stock; (4) no dark brown (v+; bw) or bright red (v; bw+) phenotypes were found in the progeny of the experimental embryos; (5) the “information” for the altered phenotype is frequently lost during gamete formation; (6) duplications of chromosomal segments including the band in a polytene chromosome corresponding to the vermilion (v) gene are observed in two stocks.Ten iso-DNA induced stocks and their progeny have the following characteristics: (1) there is no unstable period; (2) expression of the altered phenotype is intermediate; (3) in some stocks the level of expression varies from individual to individual but others show a uniform phenotype; (4) no dark brown or birght red phenotypes were found in the progeny of the experimental embryos; (5) the “information” for the altered phenotype is stably trasmitted through all gametes of a phenotypically altered fly.These results suggest that the altered phenotype in some stocks derived from allo-DNA injected embryos may have been obtained from gene transfer at the v locus, whereas the remaining stocks, both allo- and iso-DNA induced, are likely to be DNA induced mutations.