The genetic dissection of the fruitless gene's functions during embryogenesis in Drosophila melanogaster

The fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene having sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, little is known about its sex-nonspecific functions. The embryonic central nervous system (CNS) is a prime model system in which to study the genetic control of axonal outgrowth and proper CNS formation. I have examined fru's sex-nonspecific role in embryonic neural development. fru transcripts and FRU proteins from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis and subsequently in both neurons and glia. In embryos that lack most or all fru function, Fasciclin II- and BP102-positive axons appeared to defasciculate from their normal pathway and fasciculate along aberrant neuronal pathways, suggesting that one of fru's sex-nonspecific roles is to regulate axonal differentiation. I next examined whether the loss of fru function in FRU-expressing neuronal precursors causes neuronal fate change. Analysis of fru mutant embryos revealed a lack of Even-skipped (Eve) staining in Eve-expressing neurons, ectopic Eve staining in non-Eve-expressing neurons and mispositioned dorsal Eve-expressing neurons, which suggests that fru functions to maintain neuronal identity rather than to specify neuronal fate. In fru mutants these defects in axonal projections and in Eve staining were rescued by the expression of specific fru transgenes. To better understand fru's function in the formation of the embryonic CNS, I dissected out fru's function in neuron and glia through a genetic interaction study. fru genetically interacts in neurons with longitudinal lacking to make proper axonal projections. In addition, fru might be in the same genetic pathway as roundabout (robo), a repulsive guidance receptor, and commissureless, a downregulator of Robo, to ensure proper axonal pathfinding. Surprisingly, fru interacts with tramtrack and glial cells missing to repress neuronal differentiation in the lateral glia and with single-minded for the development of midline glia. Taken together, fru function is required for proper axonal pathfinding in neurons and for proper development of lateral and midline glia

Abeta42-Induced Neurodegeneration via an Age-Dependent Autophagic-Lysosomal Injury in Drosophila

The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD) remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Aβ1–42) is believed to play a causative role in the development of AD. Here we expressed human Aβ1–42 and amyloid beta 40 (Aβ1–40) in Drosophila neurons. Aβ1–42 but not Aβ1–40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Aβ1–42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Aβ1–42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Aβ1–42 and Aβ1–40, and reveal an Aβ1–42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury

The Toll→NFκB Signaling Pathway Mediates the Neuropathological Effects of the Human Alzheimer's Aβ42 Polypeptide in Drosophila

Alzheimer's (AD) is a progressive neurodegenerative disease that afflicts a significant fraction of older individuals. Although a proteolytic product of the Amyloid precursor protein, the Αβ42 polypeptide, has been directly implicated in the disease, the genes and biological pathways that are deployed during the process of Αβ42 induced neurodegeneration are not well understood and remain controversial. To identify genes and pathways that mediated Αβ42 induced neurodegeneration we took advantage of a Drosophila model for AD disease in which ectopically expressed human Αβ42 polypeptide induces cell death and tissue degeneration in the compound eye. One of the genes identified in our genetic screen is Toll (Tl). It encodes the receptor for the highly conserved Tl→NFkB innate immunity/inflammatory pathway and is a fly homolog of the mammalian Interleukin-1 (Ilk-1) receptor. We found that Tl loss-of-function mutations dominantly suppress the neuropathological effects of the Αβ42 polypeptide while gain-of-function mutations that increase receptor activity dominantly enhance them. Furthermore, we present evidence demonstrating that Tl and key downstream components of the innate immunity/inflammatory pathway play a central role in mediating the neuropathological activities of Αβ42. We show that the deleterious effects of Αβ42 can be suppressed by genetic manipulations of the Tl→NFkB pathway that downregulate signal transduction. Conversely, manipulations that upregulate signal transduction exacerbate the deleterious effects of Aβ42. Since postmortem studies have shown that the Ilk-1→NFkB innate immunity pathway is substantially upregulated in the brains of AD patients, the demonstration that the Tl→NFkB signaling actively promotes the process of Αβ42 induced cell death and tissue degeneration in flies points to possible therapeutic targets and strategies

A model for studying Alzheimer's Abeta42-induced toxicity in Drosophila melanogaster.

Alzheimer's disease is a neurological disorder resulting in the degeneration and death of brain neurons controlling memory, cognition and behavior. Although overproduction of Abeta peptides is widely considered a causative event in the disease, the mechanisms by which Abeta peptides cause neurodegeneration and the processes of Abeta clearance and degradation remain unclear. To address these issues, we have expressed the Abeta peptides in Drosophila melanogaster. We show that overexpression of Abeta42 peptides in the nervous system results in phenotypes associated with neuronal degeneration in a dose- and age-dependent manner. We further show that a mutation in a Drosophila neprilysin gene suppresses the Abeta42 phenotypes by lowering the levels of the Abeta42 peptide, supporting the role of neprilysin in the catabolism of Abeta peptides in vivo. We propose that our Drosophila model is suitable for the study and elucidation of Abeta metabolism and toxicity at the genetic level

Identification of Novel Genes That Modify Phenotypes Induced by Alzheimer's β-Amyloid Overexpression in Drosophila

Sustained increases in life expectancy have underscored the importance of managing diseases with a high incidence in late life, such as various neurodegenerative conditions. Alzheimer's disease (AD) is the most common among these, and consequently significant research effort is spent on studying it. Although a lot is known about the pathology of AD and the role of β-amyloid (Aβ) peptides, the complete network of interactions regulating Aβ metabolism and toxicity still eludes us. To address this, we have conducted genetic interaction screens using transgenic Drosophila expressing Aβ and we have identified mutations that affect Aβ metabolism and toxicity. These analyses highlight the involvement of various biochemical processes such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function, among others, in mediating toxic Aβ effects. Several of the mutations that we identified have not been linked to Aβ toxicity before and thus constitute novel potential targets for AD intervention. We additionally tested these mutations for interactions with tau and expanded-polyglutamine overexpression and found a few candidate mutations that may mediate common mechanisms of neurodegeneration. Our data offer insight into the toxicity of Aβ and open new areas for further study into AD pathogenesi

Identification of novel genes that modify phenotypes induced by Alzheimer's beta-amyloid overexpression in Drosophila.

Sustained increases in life expectancy have underscored the importance of managing diseases with a high incidence in late life, such as various neurodegenerative conditions. Alzheimer's disease (AD) is the most common among these, and consequently significant research effort is spent on studying it. Although a lot is known about the pathology of AD and the role of beta-amyloid (Abeta) peptides, the complete network of interactions regulating Abeta metabolism and toxicity still eludes us. To address this, we have conducted genetic interaction screens using transgenic Drosophila expressing Abeta and we have identified mutations that affect Abeta metabolism and toxicity. These analyses highlight the involvement of various biochemical processes such as secretion, cholesterol homeostasis, and regulation of chromatin structure and function, among others, in mediating toxic Abeta effects. Several of the mutations that we identified have not been linked to Abeta toxicity before and thus constitute novel potential targets for AD intervention. We additionally tested these mutations for interactions with tau and expanded-polyglutamine overexpression and found a few candidate mutations that may mediate common mechanisms of neurodegeneration. Our data offer insight into the toxicity of Abeta and open new areas for further study into AD pathogenesis

Dorsal mediates Aβ42 dependent neurodegeneration of the eye.

<p>Panels A–E: In the experiments shown in this figure all flies are siblings that are homozygous for the pGMR-Aβ42 transgene. The fly on the left side of each panel is a wild type sib, while the fly on the right side of each panel is a sib that is either heterozygous or homozygous for the indicated <i>dl</i> mutation. Panel A: <i>dl¹</i>/+. Panel B: <i>dl⁴</i>/+ Panel C: <i>dl⁸</i>/+ Panel D: <i>dl¹</i>/<i>dl¹</i>. Panel E: <i>dl⁴/dl⁴</i>. Panel F: In this experiment both flies are hemizygous for the pGMR-Aβ42 transgene. The fly on the left is wild type, while the fly on the right is homozygous <i>dl^`</i>. Arrows in panels A and E point to ommatidia with dead or dying cells.</p

Effect of mutations in the <i>Tl→ΝFκB</i> pathway on the Aβ42 induced rough eye phenotype.

<p>The rough eye phenotype of pGMR-Aβ42 flies was assigned a value of ++++. According to this scoring system, strong, moderate and weak suppression corresponded to +, ++, and +++. Weak and moderate enhancement corresponded to +++++ and ++++++. All flies are hemizygous for the transgene and either wild type (+) or heterozygous for the indicated mutation.</p

Components of the <i>Toll-NF</i>κ<i>B</i> signaling pathway modulate Aβ42 polypetide induced neurodegeneration.

<p>Panels A–F: In the experiments shown in this figure all flies are siblings that are hemizygous for the pGMR-Aβ42 transgene. The fly on the left side of each panel is a wild type sib, while the fly on the right side of each panel is a sib that is heterozygous for the indicated mutation in the <i>Toll-NF</i>κ<i>B</i> signaling pathway mutant. Panel A: <i>dl¹</i>. Panel B: <i>dl⁴</i>. Panel C: <i>dl⁸</i>. Panel D: <i>pll²</i>. Panel E: <i>pll⁷</i>. Panel F: <i>tub</i>.</p