Aggregation of the amyloid-β-42 (Aβ42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer's disease (AD), and the prevention of Aβ aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Aβ can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Aβ42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Aβ42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Aβ42Arc) and an artificial mutation (Aβ42art) that is known to suppress aggregation and toxicity of Aβ42 in vitro. In the Drosophila brain, Aβ42Arc formed more oligomers and deposits than did wild type Aβ42, while Aβ42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Aβ peptides. Surprisingly, however, Aβ42art caused earlier onset of memory defects than Aβ42. More remarkably, each Aβ induced qualitatively different pathologies. Aβ42Arc caused greater neuron loss than did Aβ42, while Aβ42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Aβ aggregates: Aβ42Arc formed large deposits in the cell body, Aβ42art accumulated preferentially in the neurites, while Aβ42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Aβ42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.

The amyloid-β 42 (Aβ42) is thought to play a central role in the pathogenesis of Alzheimer's disease (AD). However, the molecular mechanisms by which Aβ42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Aβ42 induces mitochondrial mislocalization, which contributes to Aβ42-induced neuronal dysfunction in a transgenic Drosophila model. In the Aβ42 fly brain, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. In contrast, organization of microtubule or global axonal transport was not significantly altered at this stage. Aβ42-induced behavioral defects were exacerbated by genetic reductions in mitochondrial transport, and were modulated by cAMP levels and PKA activity. Levels of putative PKA substrate phosphoproteins were reduced in the Aβ42 fly brains. Importantly, perturbations in mitochondrial transport in neurons were sufficient to disrupt PKA signaling and induce late-onset behavioral deficits, suggesting a mechanism whereby mitochondrial mislocalization contributes to Aβ42-induced neuronal dysfunction. These results demonstrate that mislocalization of mitochondria underlies the pathogenic effects of Aβ42 in vivo.

The amyloid-␤42 (A␤42) peptide has been suggested to play a causative role in Alzheimer disease (AD). Neprilysin (NEP) is one of the rate-limiting A␤-degrading enzymes, and its enhancement ameliorates extracellular amyloid pathology, synaptic dysfunction, and memory defects in mouse models of A␤ amyloidosis. In addition to the extracellular A␤, intraneuronal A␤42 may contribute to AD pathogenesis. However, the protective effects of neuronal NEP expression on intraneuronal A␤42 accumulation and neurodegeneration remain elusive. In contrast, sustained NEP activation may be detrimental because NEP can degrade many physiological peptides, but its consequences in the brain are not fully understood. Using transgenic Drosophila expressing human NEP and A␤42, we demonstrated that NEP efficiently suppressed the formation of intraneuronal A␤42 deposits and A␤42-induced neuron loss. However, neuronal NEP overexpression reduced cAMP-responsive element-binding protein-mediated transcription, caused age-dependent axon degeneration, and shortened the life span of the flies. Interestingly, the mRNA levels of endogenous fly NEP genes and phosphoramidon-sensitive NEP activity declined during aging in fly brains, as observed in mammals. Taken together, these data suggest both the protective and detrimental effects of chronically high NEP activity in the brain. Down-regulation of NEP activity in aging brains may be an evolutionarily conserved phenomenon, which could predispose humans to developing late-onset AD. Alzheimer disease (AD)3 is a progressive neurodegenerative disease defined by two protein deposits in autopsied brains: extracellular amyloid plaques composed of the 40-or 42-amino acid ␤-amyloid peptides (A␤40 or A␤42), and intracellular neurofibrillary tangles composed of abnormally hyperphosphorylated microtubule-associated protein Tau (1). A␤ peptides are physiological metabolites of the amyloid precursor protein (APP) resulting from sequential cleavage by ␤-secretase and ␥-secretase complex, whose catalytic subunits are presenilin 1 (PS1) and presenilin 2 (PS2) (2). Molecular genetic studies of early onset familial AD patients have identified causative mutations in APP, PS1, and PS2 genes, and these mutations promote A␤42 production, aggregation, and stability against clearance (3). Thus, the increased A␤42 levels in the brain are believed to initiate AD pathogenesis.The mechanisms by which A␤42 reaches pathological levels in the brains of late-onset AD (LOAD) patients are not well understood. The steady state level of A␤42 in the brain reflects the balance between production and clearance, and a reduction in clearance activity would raise A␤42 levels. A deficiency in neprilysin (NEP), a major rate-limiting A␤-degrading enzyme (4, 5), accelerates formation of extracellular amyloid deposits (6), amyloid angiopathy (6), synaptic dysfunctions (7), and memory defects (7) caused by human A␤ in transgenic mice. In LOAD patients, NEP mRNA and pro-

Hyperphosphorylation of the microtubule associated protein tau is detected in the brains of individuals with a range of neurodegenerative diseases including Alzheimer's disease (AD). An imbalance in phosphorylation and/or dephosphorylation of tau at disease-related sites has been suggested to initiate the abnormal metabolism and toxicity of tau in disease pathogenesis. However, the mechanisms underlying abnormal phosphorylation of tau in AD are not fully understood. Here, we show that the DNA damage-activated Checkpoint kinase 2 (Chk2) is a novel tau kinase and enhances tau toxicity in a transgenic Drosophila model. Overexpression of Drosophila Chk2 increases tau phosphorylation at Ser262 and enhances tau-induced neurodegeneration in transgenic flies expressing human tau. The non-phosphorylatable Ser262Ala mutation abolishes Chk2-induced enhancement of tau toxicity, suggesting that the Ser262 phosphorylation site is involved in the enhancement of tau toxicity by Chk2. In vitro kinase assays revealed that human Chk2 and a closely related checkpoint kinase 1 (Chk1) directly phosphorylate human tau at Ser262. We also demonstrate that Drosophila Chk2 does not modulate the activity of the fly homolog of microtubule affinity regulating kinase, which has been shown to be a physiological tau Ser262 kinase. Since accumulation of DNA damage has been detected in the brains of AD patients, our results suggest that the DNA damage-activated kinases Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in the pathogenesis of AD.

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), we have examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot with functions of adipose tissue and the liver in flies, on energy balance, stress resistance and feeding behavior. We found that disruption of CREB function in neurons reduced glycogen and lipid stores and increased sensitivity to starvation. Expression of DN-CREB in the fat body also reduced glycogen levels, while it did not affect starvation sensitivity, presumably due to increased lipid levels in these flies. Interestingly, blocking CREB activity in the fat body increased food intake. These flies did not show a significant change in overall body size, suggesting that disruption of CREB activity in the fat body caused an obese-like phenotype. Using a transgenic CRE-luciferase reporter, we further demonstrated that disruption of the adipokinetic hormone receptor, which is functionally related to mammalian glucagon and β-adrenergic signaling, in the fat body reduced CRE-mediated transcription in flies. This study demonstrates that CREB activity in either neuronal or peripheral tissues regulates energy balance in Drosophila, and that the key signaling pathway regulating CREB activity in peripheral tissue is evolutionarily conserved.