Accumulation of amyloid-␤ (A␤) peptides in the brain has been suggested to be the primary event in sequential progression of Alzheimer's disease (AD). Here, we use Drosophila to examine whether expression of either the human A␤40 or A␤42 peptide in the Drosophila brain can induce pathological phenotypes resembling AD. The expression of A␤42 led to the formation of diffused amyloid deposits, age-dependent learning defects, and extensive neurodegeneration. In contrast, expression of A␤40 caused only age-dependent learning defects but did not lead to the formation of amyloid deposits or neurodegeneration. These results strongly suggest that accumulation of A␤42 in the brain is sufficient to cause behavioral deficits and neurodegeneration. Moreover, Drosophila may serve as a model for facilitating the understanding of molecular mechanisms underlying A␤ toxicity and the discovery of novel therapeutic targets for AD. A lzheimer's disease (AD) is a neurodegenerative disorder characterized clinically by progressive decline in memory accompanied by histological changes, including neuronal loss and the formation of neurofibrillary tangles (NFTs) and senile plaques (1). The accumulation of amyloid-␤ (A␤)42 peptide, the major component of senile plaques, has been hypothesized to be the primary event in AD pathogenesis (2, 3). The strongest support for the A␤ hypothesis comes from genetic analyses of familial AD (FAD); most FAD mutations identified in A␤ precursor protein (APP), Presenilin1 (PS1) and Presenilin2 (PS2) genes appear to cause excessive accumulation of A␤42 (4). Secretion of A␤ peptides is a result of sequential cleavage of APP by ␤-secretase, a type I transmembrane glycosylated aspartyl protease, and ␥-secretase, a large protein complex that includes at least four proteins, Presenilins (PS1 or PS2), Nicastrin, Aph-1, and Pen-2 (for review, see ref. 5). The heterogeneity of ␥-secretase cleavage gives rise to a series of A␤ peptides, including the major species A␤40 and a smaller amount of A␤42.To study AD pathogenesis in vivo, a number of AD mouse models have been established and have successfully recapitulated AD-like phenotypes, including abundant amyloid deposits, astroglial activation, synaptic loss and dysfunction, behavioral abnormalities, and neurodegeneration (6-15). In addition to these mouse models, the model systems that allow highthroughput genetic screening will facilitate the discovery of genes involved in AD pathogenesis. Furthermore, one of the intriguing issues that have not been elucidated in these transgenic mice is the pathological roles of each specific A␤ species (i.e., A␤40 and A␤42), because currently available mouse models mainly rely on overexpression of APP.We use a Drosophila model (16) to compare the specific pathological roles of A␤40 and A␤42. In Drosophila, all components involved in the protein complex responsible for ␥-secretase activity are highly conserved (17), whereas ␤-secretase activity is absent or very low (18). An APP-like protein (APPL) is also present in flies, althou...

An anatomical and electrophysiological study of Drosophila mutants has been made to determine the effect of altered electrical activity on the development and maintenance of larval neuromuscular junctions. We examined motor axon terminals of (1) hyperexcitable mutants Shaker (Sh), ether a go-go (eag), Hyperkinetic (Hk), and Duplication of para+ (Dp para+); and (2) mutants with reduced excitability, no action potential (napts) and paralytic (parats 1). Nerve terminals innervating larval body-wall muscles were visualized by using anti-HRP immunocytochemistry, which specifically stains neurons in insect species. In wild-type larvae, motor axon terminals were distributed in a stereotypic fashion. However, in combinations of eag and Sh alleles, the basic pattern of innervation was altered. There was an increase in both the number of higher-order axonal branches over the muscles and the number of varicosities on the neurites. A similar phenomenon was found in the double mutant Hk eag and, to a lesser extent, in Dp para+ and Dp para+ Sh mutants. It is known that at permissive temperature the napts, but not parats 1, mutation decreases excitability of larval motor axons and suppresses the behavioral phenotypes of Sh, eag, and Hk. In the mutant napts (reared at permissive temperature), a slight decrease in the extent of branching was observed. Yet, when combined with eag Sh, napts completely reversed the morphological abnormality in eag Sh mutants. No such reversion was observed in parats 1 eag Sh mutants. The endogenous patterns of electrical activity at the neuromuscular junction were analyzed by extracellular recordings in a semi-intact larval preparation. Recordings from wild-type body-wall muscles revealed rhythmic bursts of spikes. In eag Sh mutants, this rhythmic activity was accompanied by or superimposed on periods of strong tonic activity. This abnormal pattern of activity could be partially suppressed by napts in combination with eag Sh.

Initially acquired memory dissipates rapidly if not consolidated. Such memory decay is thought to result either from the inherently labile nature of newly acquired memories or from interference by subsequently attained information. Here we report that a small G protein Rac-dependent forgetting mechanism contributes to both passive memory decay and interference-induced forgetting in Drosophila. Inhibition of Rac activity leads to slower decay of early memory, extending it from a few hours to more than one day, and to blockade of interference-induced forgetting. Conversely, elevated Rac activity in mushroom body neurons accelerates memory decay. This forgetting mechanism does not affect memory acquisition and is independent of Rutabaga adenylyl cyclase-mediated memory formation mechanisms. Endogenous Rac activation is evoked on different time scales during gradual memory loss in passive decay and during acute memory removal in reversal learning. We suggest that Rac's role in actin cytoskeleton remodeling may contribute to memory erasure.

Pioneering research studies, beginning with those using Drosophila, have identified several molecular and cellular mechanisms for active forgetting. The currently known mechanisms for active forgetting include neurogenesis-based forgetting, interference-based forgetting, and intrinsic forgetting, the latter term describing the brain’s chronic signaling systems that function to slowly degrade molecular and cellular memory traces. The best-characterized pathway for intrinsic forgetting includes “forgetting cells” that release dopamine onto engram cells, mobilizing a signaling pathway that terminates in the activation of Rac1/cofilin to effect changes in the actin cytoskeleton and neuron/synapse structure. Intrinsic forgetting may be the default state of the brain, constantly promoting memory erasure and competing with processes that promote memory stability like consolidation. A better understanding of active forgetting will provide insights into the brain’s memory management system and human brain disorders that alter active forgetting mechanisms.

Activity-dependent synaptic plasticity has been implicated in the refinement and modification of neural circuits during development and learning. Previous studies show that activity-induced facilitation and potentiation are disrupted at larval neuromuscular junctions in the memory mutants dunce (dnc) and rutabaga (rut) of Drosophila. The diminished learning-memory capacity and synaptic transmission plasticity have been associated with altered cAMP levels since dnc affects the cAMP-specific phosphodiesterase and rut affects adenylate cyclase. In this study, the morphology of larval motor axon terminals was examined by anti-HRP immunohistochemistry. It was found that the numbers of terminal varicosities and branches were increased in dnc mutants, which have elevated cAMP concentrations. Such increase was suppressed in dnc rut double mutants by rut mutations, which reduce cAMP synthesis. More profuse projections of larval motor axons have also been reported in double-mutant combinations of ether à go-go (eag) and Shaker (Sh) alleles, which display greatly enhanced nerve activity as a result of reduction in different K+ currents. Therefore, we examined combinations of dnc and rut with eag and Sh mutations to explore the possible relation between activity- and cAMP-induced morphological changes. We found that the expanded projections in dnc were further enhanced in double mutants of dnc with either eag or Sh, an effect that could again be suppressed by rut. The results provide evidence for altered plasticity of synaptic morphology in memory mutants dnc and rut and suggest a role of cAMP cascade in mediating activity-dependent synaptic plasticity.

Synaptic transmission was examined in Drosophila mutants deficient in memory function. These mutants, dunce and rutabaga, are defective in different steps of the cyclic adenosine 3',5'-monophosphate (cAMP) cascade. In both dunce and rutabaga larvae, voltage-clamp analysis of neuromuscular transmission revealed impaired synaptic facilitation and post-tetanic potentiation as well as abnormal responses to direct application of dibutyryl cAMP. In addition, the calcium dependence of transmitter release was shifted in dunce. The results suggest that the cAMP cascade plays a role in synaptic facilitation and potentiation and indicate that synaptic plasticity is altered in Drosophila memory mutants.

The tumour-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP). In addition to being involved in tumour formation, NF1 has been reported to cause learning defects in humans and Nf1 knockout mice. However, it remains to be determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing 60% identity of its 2,803 amino acids with human NF1 (ref. 12). Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase. Because rut was isolated as a learning and short-term memory mutant, we have pursued the hypothesis that NF1 may affect learning through its control of the Rut-adenylyl cyclase/cAMP pathway. Here we show that NF1 affects learning and short-term memory independently of its developmental effects. We show that G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and that the mechanism of the NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory.

The neurofibromatosis type 1 (NF1) tumor suppressor protein is thought to restrict cell proliferation by functioning as a Ras-specific guanosine triphosphatase-activating protein. However, Drosophila homozygous for null mutations of an NF1 homolog showed no obvious signs of perturbed Ras1-mediated signaling. Loss of NF1 resulted in a reduction in size of larvae, pupae, and adults. This size defect was not modified by manipulating Ras1 signaling but was restored by expression of activated adenosine 3', 5'-monophosphate-dependent protein kinase (PKA). Thus, NF1 and PKA appear to interact in a pathway that controls the overall growth of Drosophila.