Role of ATP-dependent calcium regulation in modulation of Drosophila synaptic thermotolerance.

Maintenance of synaptic transmission requires regulation of intracellular Ca(2+) in presynaptic nerve terminals; loss of this regulation at elevated temperatures may cause synaptic failure. Accordingly, we examined the thermosensitivity of presynaptic calcium regulation in Drosophila larval neuromuscular junctions, testing for effects of disrupting calcium clearance. Motor neurons were loaded with the ratiometric Ca(2+) indicator Fura-dextran to monitor calcium regulation as temperature increased. Block of the Na(+)/Ca(2+) exchanger or removal of extracellular Ca(2+) prevented the normal temperature-induced increase in resting calcium. Conversely, two treatments that interfered with Ca(2+) clearance-inactivation of the endoplasmic reticulum Ca(2+)-ATPase with thapsigargin and inhibition of the plasma membrane Ca(2+)-ATPase with high pH-significantly accelerated the temperature-induced rise in resting Ca(2+) concentration and reduced the thermotolerance of synaptic transmission. Disrupting Ca(2+)-ATPase function by interfering with energy production also facilitated the temperature-induced rise in resting [Ca(2+)] and reduced thermotolerance of synaptic transmission. Conversely, fortifying energy levels with extra intracellular ATP extended the operating temperature range of both synaptic transmission and Ca(2+) regulation. In each of these cases, Ca(2+) elevations evoked by an electrical stimulation of the nerve (evoked Ca(2+) responses) failed when resting Ca(2+) remained >e 200 nM for several minutes. Failure of synaptic function was correlated with the release of intracellular calcium stores, and we provide evidence suggesting that release from the mitochondria disrupts evoked calcium responses and synaptic transmission. Thus the thermal limit of synaptic transmission may be directly linked to the stability of ATP-dependent mechanisms that regulate intracellular ion concentrations in the nerve terminal.

Demented flies? Using Drosophila to model human neurodegenerative diseases.

The success of biomedical research in the past few decades has led to dramatic improvements in human health and, as a result, increased life expectancy. An unexpected consequence, however, has been an increase in the number of age-related diseases and, in particular, neurodegenerative diseases. Despite their prevalence, a therapeutic void exists in part due to an incomplete understanding of the biochemical pathogenesis of these diseases. A powerful method that can be used to understand the basic mechanisms underlying neurodegenerative diseases is to generate animal models based on manipulating the expression of single genes that are disease causative. This approach has been facilitated by the fact that many neurodegenerative diseases are inherited as autosomal dominant traits such that expression of the mutant gene in a model organism might be expected to recapitulate the disease. During the past few years, the fruit fly, Drosophila melanogaster, has emerged as a powerful tool to model human neurodegenerative diseases. Here, we describe the various approaches utilized to create fly models of human neurodegenerative disease, and how they can aid in our understanding of disease pathogenesis and facilitate drug discovery and testing.

Identification of a novel family of putative methyltransferases that interact with human and Drosophila presenilins.

Mutations in the presenilin genes have been shown to cause the majority of cases of early-onset familial Alzheimer's disease (AD). In addition to their role in AD, presenilins are also known to function during development by interacting with the Notch pathway. To determine if presenilins have additional functions during development and AD we have used a yeast two-hybrid approach to search for proteins that can bind to presenilins. Here, we show the identification and characterization of a novel putative methyltransferase (Metl) that interacts with the loop region of Drosophila presenilin as well as human presenilin-1 and presenilin-2, suggesting that this interaction is evolutionarily conserved and functionally important. Metl appears to be a member of a conserved family of methyltransferases that share homology with, but are distinct from, the UbiE family of methyltransferases involved in ubiquinone and menaquinone biosynthesis. In Drosophila, the metl gene gives rise to two major isoforms by alternative splicing that are broadly expressed throughout development and found in the central nervous system in an overlapping pattern with Drosophila presenilin. Finally, we show that two independent dominant adult phenotypes produced by overexpression of presenilin can be enhanced by overexpression of metl in the same tissue. Taken together, these results suggest that presenilin and Metl functionally and genetically interact during development.

Drosophila Amphiphysin is a post-synaptic protein required for normal locomotion but not endocytosis.

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin-mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.

Neuralized functions as an E3 ubiquitin ligase during Drosophila development.

The Notch pathway is a widely studied means of intercellular signaling responsible for the determination of cell fate, cell differentiation, and boundary formation (reviewed in ). The main effectors of this pathway, Notch (N) and Delta (Dl), have been shown to function as a receptor and ligand, respectively. Genetic and phenotypic studies suggest that Neuralized (Neu), a RING finger protein, also plays a role within the N-Dl pathway, although its biochemical function is unknown. Here, we show that Neu is required at the plasma membrane for functional activity and that its RING finger domain acts as an E3 ubiquitin ligase. These data suggest that the role of Neu is to target components of the N-Dl pathway for ubiquitination, allowing for propagation and/or regulation of the signal.

Analysis of the mutant Drosophila N-ethylmaleimide sensitive fusion-1 protein in comatose reveals molecular correlates of the behavioural paralysis.

NEM-sensitive fusion protein (NSF) is an ATPase required for many intracellular membrane trafficking steps. Recent studies have suggested that NSF alters the conformation of the SNAP receptors (SNAREs) to permit their interaction, or to uncouple them after they interact. Most organisms have a single NSF gene product but Drosophila express two highly related isoforms, dNSF-1 and dNSF-2. dNSF-1 is encoded by the gene comatose (comt), first identified as the locus of a temperature-sensitive paralytic mutation. Here we show that dNSF-1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation revealed that dNSF-1 was enriched in a vesicle fraction along with the synaptic vesicle protein synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlated with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comt(TP7) larvae, there is no associated neurophysiological phenotype at the neuromuscular junction (nmj) or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but that in the larval nmj function may be maintained by other isoforms.

SNARE-dependent signaling at the Drosophila wing margin.

The wing of Drosophila melanogaster has long been used as a model system to characterize intermolecular interactions important in development. Implicit in our understanding of developmental processes is the proper trafficking and sorting of signaling molecules, although the precise mechanisms that regulate membrane trafficking in a developmental context are not well studied. We have therefore chosen the Drosophila wing to assess the importance of SNARE-dependent membrane trafficking during development. N-Ethylmaleimide-sensitive fusion protein (NSF) is a key component of the membrane-trafficking machinery and we constructed a mutant form of NSF whose expression we directed to the developing wing margin. This resulted in a notched-wing phenotype, the severity of which was enhanced when combined with mutants of VAMP/Synaptobrevin or Syntaxin, indicating that it results from impaired membrane trafficking. Importantly, we find that the phenotype is also enhanced by mutations in genes for wingless and components of the Notch signaling pathway, suggesting that these signaling pathways were disrupted. Finally, we used this phenotype to conduct a screen for interacting genes, uncovering two Notch pathway components that had not previously been linked to wing development. We conclude that SNARE-mediated membrane trafficking is an important component of wing margin development and that dosage-sensitive developmental pathways will act as a sensitive reporter of partial membrane-trafficking disruption.

Neuronal regulation of lifespan: clues from flies and worms.

Aging is a universal but poorly understood biological process that involves a complex interplay between environmental and genetic factors. Model organisms such as C. elegans and Drosophila have provided an opportunity to search for and identify specific genes that affect life span. Interestingly, increasing evidence suggests that many of these genes affect lifespan by exerting their effects in a limited number of cell types and, in particular, neurons, indicating that the nervous system may be a central regulator of organismal lifespan.

SNARE proteins contribute to calcium cooperativity of synaptic transmission.

A hallmark of calcium-triggered synaptic transmission is the cooperative relationship between calcium and the amount of transmitter released. This relationship is thought to be important for improving the efficiency of synaptic vesicle exocytosis. Although it is generally held that cooperativity arises from the interaction of multiple calcium ions with a single calcium-sensing molecule, the precise molecular basis of this phenomenon is not known. The SNARE proteins are known to be critical for synaptic vesicle exocytosis. We therefore tested for a contribution of SNARE proteins to cooperativity by genetically reducing the levels of syntaxin IA and neuronal-synaptobrevin in Drosophila. Surprisingly, we found that reducing these SNARE proteins also reduced Ca(2+) cooperativity. Thus, SNARE proteins are important for determining the cooperative relationship between calcium and synaptic transmission.

Neuralized functions cell autonomously to regulate Drosophila sense organ development.

Neurogenic genes, including Notch and Delta, are thought to play important roles in regulating cell-cell interactions required for Drosophila sense organ development. To define the requirement of the neurogenic gene neuralized (neu) in this process, two independent neu alleles were used to generate mutant clones. We find that neu is required for determination of cell fates within the proneural cluster and that cells mutant for neu autonomously adopt neural fates when adjacent to wild-type cells. Furthermore, neu is required within the sense organ lineage to determine the fates of daughter cells and accessory cells. To gain insight into the mechanism by which neu functions, we used the GAL4/UAS system to express wild-type and epitope-tagged neu constructs. We show that Neu protein is localized primarily at the plasma membrane. We propose that the function of neu in sense organ development is to affect the ability of cells to receive Notch-Delta signals and thus modulate neurogenic activity that allows for the specification of non-neuronal cell fates in the sense organ.

Physical and genetic interaction of filamin with presenilin in Drosophila.

Presenilins were first identified as causative factors in early onset, familial Alzheimer's Disease (FAD). They are predicted to encode a highly conserved novel family of eight transmembrane domain proteins with a large hydrophilic loop between TM6 and TM7 that is the site of numerous FAD mutations. Here, we show that the loop region of Drosophila and human presenilins interacts with the C-terminal domain of Drosophila filamin. Furthermore, we show that Drosophila has at least two major filamin forms generated by alternative splicing from a gene that maps to position 89E10-89F4 on chromosome 3. The longest form is enriched in the central nervous system and ovaries, shares 41.7% overall amino acid identity with human filamin (ABP-280) and contains an N-terminal actin-binding domain. The shorter form is broadly expressed and encodes an alternatively spliced form of the protein lacking the actin-binding domain. Finally, we show that presenilin and filamin are expressed in overlapping patterns in Drosophila and that dominant adult phenotypes produced by overexpression of presenilin can be suppressed by overexpression of filamin in the same tissue. Taken together, these results suggest that presenilin and filamin functionally interact during development.

Drosophila presenilin is required for neuronal differentiation and affects notch subcellular localization and signaling.

Presenilins are a highly conserved family of proteins first identified as causative genes in early onset familial Alzheimer's disease. Recent studies have suggested a role for presenilins in the Notch-signaling pathway, but their specific function within this pathway remains unclear. Here, we have characterized the Drosophila presenilin gene and protein and studied their interaction with Notch in both mutants and transgenics. We find that the Drosophila presenilin protein is proteolytically cleaved and broadly expressed during development with the highest levels in neurons within the larval CNS. We also show that mutations in Drosophila presenilin (Dps) genetically interact with Notch and result in an early pupal-lethal phenotype characterized by defects in eye and wing development and incomplete neuronal differentiation within the larval CNS. Moreover, we find that processing of Notch in the Golgi by the furin protease is unaffected in Dps mutants and that Notch is present and may even accumulate on the plasma membrane of neuroblasts in the larval CNS of Dps mutants. In contrast, overexpression of Dps in transgenics causes Notch to accumulate in the cytoplasm. Taken together, these results indicate that Drosophila presenilin is required for proper neuronal differentiation and may regulate the subcellular localization of Notch proteins within cells, necessary for their accumulation and subsequent signaling capabilities.

Drosophila larval neuromuscular junction's responses to reduction of cAMP in the nervous system.

We investigated the effects of chronically lowered cyclic adenosine monophosphate (cAMP) on the morphology and physiology of the Drosophila larval neuromuscular junction, using two fly lines in which cAMP was significantly lower than normal in the nervous system: (a) transgenic flies in which the dunce (dnc) gene product was overexpressed in the nervous system, and (b) flies mutant for the rutabaga gene (rut1) which have reduced adenylyl cyclase activity. In comparison with controls, larvae with reduced cAMP exhibited a smaller number of synaptic varicosities. This effect was more pronounced in transgenic larvae, in which the reduction of neural cAMP was more pronounced. Synaptic transmission was also reduced in both cases, as evidenced by smaller excitatory junctional potentials (EJPs). Synaptic currents recorded from individual synaptic varicosities of the neuromuscular junction indicated almost normal transmitter release properties in transgenic larvae and a modest impairment in rut1 larvae. Thus, reduction in EJP amplitude in transgenic larvae is primarily due to reduced innervation, while in rut1 larvae it is attributable to the combined effects of reduced innervation and a mild impairment of transmitter release. We conclude that the major effect of chronically lowered cAMP is reduction of innervation rather than impairment of transmitter release properties.

Expression of human FALS SOD in motorneurons of Drosophila.

Mutations in human CuZn superoxide dismutase (SOD) have been associated with familial amyotrophic lateral sclerosis (FALS). Although leading to many experimental advances, this finding has not yet led to a clear understanding of the biochemical mechanism by which mutations in SOD promote the degeneration of motorneurons that causes this incurable paralytic disease. To explore the biochemical mechanism of FALS SOD-mediated neuropathogenesis, we used transgenic methodology to target the expression of a human FALS SOD to motorneurons of Drosophila, an organism known for its phenotypic sensitivity to genetic manipulation of SOD. Earlier, we showed that targeted expression of human SOD in motorneurons of Drosophila causes a dramatic extension of adult lifespan (>40%) and rescues most of the phenotypes of SOD-null mutants. Using the same genetic system, we now ask if targeted expression of a mutant allele of human SOD that is associated with FALS causes paralysis and premature death, or is otherwise injurious in Drosophila as it is in humans and transgenic mice. Here we report that high-level expression of a human FALS SOD in motorneurons is not detrimental and does not promote paralysis and premature death when expressed in motorneurons of Drosophila. In sharp contrast, the expression of FALS SOD in Drosophila actually extends lifespan, augments resistance to oxidative stress and partially rescues SOD-null mutants in a manner predicted by our earlier studies on the expression of wildtype human SOD in Drosophila motorneurons.

Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons.

Reactive oxygen (RO) has been identified as an important effector in ageing and lifespan determination. The specific cell types, however, in which oxidative damage acts to limit lifespan of the whole organism have not been explicitly identified. The association between mutations in the gene encoding the oxygen radical metabolizing enzyme CuZn superoxide dismutase (SOD1) and loss of motorneurons in the brain and spinal cord that occurs in the life-shortening paralytic disease, Familial Amyotrophic Lateral Sclerosis (FALS; ref. 4), suggests that chronic and unrepaired oxidative damage occurring specifically in motor neurons could be a critical causative factor in ageing. To test this hypothesis, we generated transgenic Drosophila which express human SOD1 specifically in adult motorneurons. We show that overexpression of a single gene, SOD1, in a single cell type, the motorneuron, extends normal lifespan by up to 40% and rescues the lifespan of a short-lived Sod null mutant. Elevated resistance to oxidative stress suggests that the lifespan extension observed in these flies is due to enhanced RO metabolism. These results show that SOD activity in motorneurons is an important factor in ageing and lifespan determination in Drosophila.

Cingulate cortex synaptic terminal proteins and neural cell adhesion molecule in schizophrenia.

The neuronal organization and patterns of afferent innervation are abnormal in the cingulate cortex in schizophrenia, and associated changes in synaptic terminals could be present. A panel of monoclonal antibodies was defined with biochemical and fusion protein studies as detecting syntaxin (antibody SP6), synaptophysin (antibody SP4) and synaptosomal-associated protein-25 (antibody SP12). These antibodies and a polyclonal antibody reactive with neural cell adhesion molecule were used to investigate the cingulate cortex in schizophrenia. Immunocytochemistry indicated that syntaxin immunoreactivity had a considerably wider distribution than synaptophysin. Overall, multivariate analysis indicated increased synaptic terminal protein immunoreactivity in schizophrenia compared to controls (P=0.004). Controlled for age and post mortem interval, syntaxin immunoreactivity was significantly elevated in schizophrenia (P=0.004), and neural cell adhesion molecule immunoreactivity was also elevated (P=0.05). The neural cell adhesion molecule to synaptophysin ratio was increased (P=0.005), possibly indicating the presence of less mature synapses in schizophrenia. Elevated syntaxin immunoreactivity is consistent with increased glutamatergic afferents to the cingulate cortex in schizophrenia, and combined with the neural cell adhesion molecule to synaptophysin ratio results suggests that synaptic function in this region in schizophrenia may be abnormal.

The manganese superoxide dismutase gene of Drosophila: structure, expression, and evidence for regulation by MAP kinase.

The gene encoding manganese superoxide dismutase (MnSOD) from Drosophila melanogaster has been isolated and its expression has been studied. In contrast to several mammalian MnSOD genes, the Drosophila gene contains a single intron and is transcribed into a single 0.8-kb transcript. Whole-mount in situ hybridization reveals extensive transcript accumulation in ovarian nurse cells and a heavy maternal contribution to the early embryo. Larval imaginal discs are enriched with MnSOD transcripts relative to other larval tissues, further suggesting a possible relationship between high MnSOD expression and mitotic activity. The 5'-upstream region contains several well-known regulatory elements including metal response, antioxidant response, and xenobiotic response elements (MRE, ARE, and XRE, respectively), sites for activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), adenosine 3',5'-cyclic monophosphate regulator binding element factor (CREB), as well as classic TATA and CAAT boxes. That MnSOD expression in Drosophila is regulated in part by the transcription factor AP-1 via the MAP kinase signal transduction pathway is suggested by experiments which show that a hypomorphic mutation of the MAP kinase-encoding rolled gene substantially reduces levels of MnSOD transcripts and correlates with reduced resistance to oxidative stress in rolled mutants.

Cloning and characterization of the Drosophila presenilin homologue.

Mutations in two genes, PS1 and PS2, coding for the presenilins, have been linked to the early onset form of familial Alzheimer's disease (AD). Here we report the identification of a Drosophila melanogaster homologue of human PS genes, Dps, which maps to band 77B-C on chromosome 3 and is expressed at multiple developmental stages. The predicted amino acid sequence of the Dps product is 53% identical to human presenilins, with the greatest similarity in the putative transmembrane domains, the hydrophobic domains at the beginning and the end of the cytoplasmic TM6-TM7 loop and the C-terminus. Analysis of Dps in a genetically tractable model system such as Drosophila may provide insight into the mechanisms of Alzheimer's disease (AD) necessary for the development of rational therapeutic approaches.

Distinct expression patterns detected within individual tissues by the GAL4 enhancer trap technique.

To identify genes that are expressed in specific cell types or tissues during development, we generated enhancer-trap lines in which the yeast transcriptional activator, GAL4, was mobilized throughout the Drosophila genome. The GAL4 lines are part of a two-part system involving GAL4 and its target, the upstream activating sequence (UAS). Detection of GAL4 expression patterns was achieved by crossing individual GAL4 lines with flies carrying the reporter gene lacZ under the transcriptional control of the UAS followed by histochemical and immunocytochemical staining. Here, we present the results of this screen and the characterization of GAL4 lines that show distinct patterns of gene expression during Drosophila development, including embryogenesis, oogenesis, and imaginal disc development. However, we were unable to identify GAL4 lines that were expressed within the germ line or during early embryogenesis. Furthermore, consistent with previous results, we found that the GAL4 enhancer trap technique had a much lower frequency of transposition than has been reported for lacZ enhancer trap screens. Taken together, these results demonstrate both the strengths and weaknesses of the GAL4 enhancer trap technique for identifying unique patterns of gene expression during development.

Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium.

Mutations in Cu/Zn superoxide dismutase (SOD), a hallmark of familial amyotrophic lateral sclerosis (FALS) in humans, are shown here to confer striking neuropathology in Drosophila. Heterozygotes with one wild-type and one deleted SOD allele retain the expected 50% of normal activity for this dimeric enzyme. However, heterozygotes with one wild-type and one missense SOD allele show lesser SOD activities, ranging from 37% for a heterozygote carrying a missense mutation predicted from structural models to destabilize the dimer interface, to an average of 13% for several heterozygotes carrying missense mutations predicted to destabilize the subunit fold. Genetic and biochemical evidence suggests a model of dimer dysequilibrium whereby SOD activity in missense heterozygotes is reduced through entrapment of wild-type subunits into unstable or enzymatically inactive heterodimers. This dramatic impairment of the activity of wild-type subunits in vivo has implications for our understanding of FALS and for possible therapeutic strategies.