Although abnormal processing of beta-amyloid precursor protein (APP) has been implicated in the pathogenic cascade leading to Alzheimer's disease, the normal function of this protein is poorly understood. To gain insight into APP function, we used a molecular-genetic approach to manipulate the structure and levels of the Drosophila APP homolog APPL. Wild-type and mutant forms of APPL were expressed in motoneurons to determine the effect of APPL at the neuromuscular junction (NMJ). We show that APPL was transported to motor axons and that its overexpression caused a dramatic increase in synaptic bouton number and changes in synapse structure. In an Appl null mutant, a decrease in the number of boutons was found. Examination of NMJs in larvae overexpressing APPL revealed that the extra boutons had normal synaptic components and thus were likely to form functional synaptic contacts. Deletion analysis demonstrated that APPL sequences responsible for synaptic alteration reside in the cytoplasmic domain, at the internalization sequence GYENPTY and a putative G(o)-protein binding site. To determine the likely mechanisms underlying APPL-dependent synapse formation, hyperexcitable mutants, which also alter synaptic growth at the NMJ, were examined. These mutants with elevated neuronal activity changed the distribution of APPL at synapses and partially suppressed APPL-dependent synapse formation. We propose a model by which APPL, in conjunction with activity-dependent mechanisms, regulates synaptic structure and number.

The vertebrate Apolipoprotein D (ApoD) is a lipocalin secreted from subsets of neurons and glia during neural development and aging . A strong correlation exists between ApoD overexpression and numerous nervous system pathologies as well as obesity, diabetes, and many forms of cancer . However, the exact relationship between the function of ApoD and the pathophysiology of these diseases is still unknown. We have generated loss-of-function Drosophila mutants for the Glial Lazarillo (GLaz) gene , a homolog of ApoD in the fruit fly, mainly expressed in subsets of adult glial cells. The absence of GLaz reduces the organism's resistance to oxidative stress and starvation and shortens male lifespan. The mutant flies exhibit a smaller body mass due to a lower amount of neutral lipids stored in the fat body. Apoptotic neural cell death increases in aged flies or upon paraquat treatment, which also impairs neural function as assessed by behavioral tests. The higher sensitivity to oxidative stress and starvation and the reduced fat storage revert to control levels when a GFP-GLaz fusion protein is expressed under the control of the GLaz natural promoter. Finally, GLaz mutants have a higher concentration of lipid peroxidation products, pointing to a lipid peroxidation protection or scavenging as the mechanism of action for this lipocalin. In agreement with Walker et al. (, in this issue of Current Biology), who analyze the effects of overexpressing GLaz, we conclude that GLaz has a protective role in stress situations and that its absence reduces lifespan and accelerates neurodegeneration.

The two pathological hallmarks of Alzheimer's disease, amyloid plaques and neurofibrillary tangles, involve two apparently unrelated proteins, the amyloid precursor protein (APP) and Tau. Although it is known that aberrant processing of APP is associated with Alzheimer's disease, the definitive role of APP in neurons is not yet clear. Tau regulates microtubule stabilization and assembly in axons and is, thus, an essential component of the microtubule-associated organelle transport machinery. Although several groups have reported physical interaction between APP and Tau, and induction of Tau phosphorylation by APP and beta-amyloid peptide, the functional connection between APP and Tau is unclear. To explore the possibility that the functions of these two proteins may somehow converge on the same cellular process, we overexpressed APPL, the Drosophila homologue of APP, along with Tau in Drosophila neurons. Panneural coexpression of APPL and Tau resulted in adults that, upon eclosion, failed to expand wings and harden the cuticle, which is suggestive of neuroendocrine dysfunction. We analyzed axonal transport when Tau and APPL were coexpressed and found that transport of axonal cargo was disrupted, as evidenced by increased retention of synaptic proteins in axons and scarcity of neuropeptide-containing vesicles in the distal processes of peptidergic neurons. In an independent approach, we demonstrated genetic interaction and phenotypic similarity between APPL overexpression and mutations in the Kinesin heavy chain (Khc) gene, the product of which is a motor for anterograde vesicle trafficking.

The Drosophila Appl gene encodes a transmembrane protein that is expressed exclusively in neurons. Amino acid comparisons show that APPL protein is a member of the amyloid precursor protein (APP)-like family of proteins. Similar to mammalian APP-family proteins, APPL is synthesized as a transmembrane holoprotein and cleaved to release a large secreted amino-terminal domain. Using immunocytochemical methods, we have analyzed the distribution of APPL in the Drosophila CNS. Surprisingly, although APPL is present in all neuronal cell bodies, the neurophil shows sterotypic differential distribution. Double-labeling experiments with different neuronal markers were used to distinguish between APPL associated with neuronal processes or extracellular matrix. The distribution of APPL protein produced from transgenes encoding wild-type (APPL), secretion-defective (APPLsd), and constitutively secreted (APPLs) forms was analyzed in an Appl-deficient background to determine which APPL form is associated with different neuropil regions. We found that APPLsd protein is enriched where APPL immunoreactivity coincides with neuronal processes. In contrast, APPLs preferentially localizes to those parts of the neuropil that show a diffuse APPL signal that rarely colocalizes with processes, and thus seems to be a component of the extracellular matrix. These data indicate that proteolytic cleavage and trafficking of APPL is differentially regulated in different neuronal populations. Through metamorphosis, APPL is especially abundant in growing axons and in areas where synapses are forming. Interestingly, in adult brains, APPL protein is enriched in the mushroom bodies and to a lesser extent in the central complex, structures involved in learning and memory.

The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6al, 6a2, 6bl, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6bl, resulting in the failure to produce all exon 6bl-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.We have reported that the haplolethal (HL) region of the Shaker gene complex (ShC) of Drosophila melanogaster (13) harbors the structural gene for troponin I (TnI) (2) and ascribed the heldup (hdp) mutations, originally described by Deak (10), to the TnI gene on the basis of genetic complementation tests. Later, Beall and Fyrberg (4) showed that some hdp mutants lack several TnI isoforms. However, since the mutant phenotype consists in the severe depletion of selected muscles, it is not clear whether the lack of specific TnI isoforms is the cause or the consequence of the muscle defect.We find that the allele mutation hdp3 consists of a single nucleotide change at the A(G/G) 3' border of the intron preceding exon 6bl of TnI, which is converted to A(A/G). The splicing complex in the mutant does not seem to recognize the new splice site and consequently does not express exon 6bl-containing RNA isoforms in any muscle, including those that do not show the mutant phenotype. The muscles not affected by the absence of exon 6bl are those in which the expression of this exon would have been quantitatively minor.TpI is a constituent of the thin filaments and plays a regulatory role in muscle contraction (33). It binds to actin, blocking the actin-myosin interaction in the resting state.Upon Ca2' entry, TnI shifts to interact with troponin C, leaving actin free and allowing the relative displacement of thin and thick filaments. Muscle physiology in insects shows peculiar features which sustain their extraordinary performance during wing beat (25). In the insect fibrillar flight muscles (DLM and DVM), calcium alone is not enough to produce full activation. The Ca2+-activated muscles are further activated in both tension and ATPase activity by small strains (1 to 3%) of their fibers. This property, first shown in the giant waterbug Lethocerus sp. (32), is also seen in D. melanogaster (23) and enables the wings to beat at high frequency. It is expected that the diversity of mechanical * Corresponding author. t Present address: