Oorvashi Roy Puli

Activation of JNK Signaling Mediates Amyloid-ß- Dependent Cell Death

Background Alzheimers disease (AD) is an age related progressive neurodegenerative disorder. One of the reasons for Alzheimers neuropathology is the generation of large aggregates of Aß42 that are toxic in nature and induce oxidative stress, aberrant signaling and many other cellular alterations that trigger neuronal cell death. However, the exact mechanisms leading to cell death are not clearly understood. Methodology/Principal Findings We employed a Drosophila eye model of AD to study how Aß42 causes cell death. Misexpression of higher levels of Aß42 in the differentiating photoreceptors of fly retina rapidly induced aberrant cellular phenotypes and cell death. We found that blocking caspase-dependent cell death initially blocked cell death but did not lead to a significant rescue in the adult eye. However, blocking the levels of c-Jun NH (2)-terminal kinase (JNK) signaling pathway significantly rescued the neurodegeneration phenotype of Aß42 misexpression both in eye imaginal disc as well as the adult eye. Misexpression of Aß42 induced transcriptional upregulation of puckered (puc), a downstream target and functional read out of JNK signaling. Moreover, a three-fold increase in phospho-Jun (activated Jun) protein levels was seen in Aß42 retina as compared to the wild-type retina. When we blocked both caspases and JNK signaling simultaneously in the fly retina, the rescue of the neurodegenerative phenotype is comparable to that caused by blocking JNK signaling pathway alone. Conclusions/Significance Our data suggests that (i) accumulation of Aß42 plaques induces JNK signaling in neurons and (ii) induction of JNK contributes to Aß42 mediated cell death. Therefore, inappropriate JNK activation may indeed be relevant to the AD neuropathology, thus making JNK a key target for AD therapies.

A Glimpse into Dorso-Ventral Patterning of the Drosophila Eye

During organogenesis in all multi‐cellular organisms, axial patterning is required to transform a single layer organ primordium into a three‐dimensional organ. The Drosophila eye model serves as an excellent model to study axial patterning. Dorso‐ventral (DV) axis determination is the first lineage restriction event during axial patterning of the Drosophila eye. The early Drosophila eye primordium has a default ventral fate, and the dorsal eye fate is established by onset of dorsal selector gene pannier (pnr) expression in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments called the equator is the site for Notch (N) activation, which triggers cell proliferation and differentiation. This review will focus on (1) chronology of events during DV axis determination; (2) how early division of eye into dorsal and ventral compartments contributes towards the growth and patterning of the fly retina, and (3) functions of DV patterning genes. Developmental Dynamics 241:69–84, 2012.

Novel Neuroprotective Function of Apical-Basal Polarity Gene Crumbs in Amyloid Beta 42 (Aβ42) Mediated Neurodegeneration

Alzheimers disease (AD, OMIM: 104300), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). We have developed a transgenic Drosophila eye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb) as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. We demonstrate a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration.

Drosophila Eye Model to Study Neuroprotective Role of CREB Binding Protein (CBP) in Alzheimer’s Disease

Background The progressive neurodegenerative disorder Alzheimer’s disease (AD) manifests as loss of cognitive functions, and finally leads to death of the affected individual. AD may result from accumulation of amyloid plaques. These amyloid plaques comprising of amyloid-beta 42 (Aβ42) polypeptides results from the improper cleavage of amyloid precursor protein (APP) in the brain. The Aβ42 plaques have been shown to disrupt the normal cellular processes and thereby trigger abnormal signaling which results in the death of neurons. However, the molecular-genetic mechanism(s) responsible for Aβ42 mediated neurodegeneration is yet to be fully understood. Methodology/Principal Findings We have utilized Gal4/UAS system to develop a transgenic fruit fly model for Aβ42 mediated neurodegeneration. Targeted misexpression of human Aβ42 in the differentiating photoreceptor neurons of the developing eye of transgenic fly triggers neurodegeneration. This progressive neurodegenerative phenotype resembles Alzheimer’s like neuropathology. We identified a histone acetylase, CREB Binding Protein (CBP), as a genetic modifier of Aβ42 mediated neurodegeneration. Targeted misexpression of CBP along with Aβ42 in the differentiating retina can significantly rescue neurodegeneration. We found that gain-of-function of CBP rescues Aβ42 mediated neurodegeneration by blocking cell death. Misexpression of Aβ42 affects the targeting of axons from retina to the brain but misexpression of full length CBP along with Aβ42 can restore this defect. The CBP protein has multiple domains and is known to interact with many different proteins. Our structure function analysis using truncated constructs lacking one or more domains of CBP protein, in transgenic flies revealed that Bromo, HAT and polyglutamine (BHQ) domains together are required for the neuroprotective function of CBP. This BHQ domain of CBP has not been attributed to promote survival in any other neurodegenerative disorders. Conclusions/Significance We have identified CBP as a genetic modifier of Aβ42 mediated neurodegeneration. Furthermore, we have identified BHQ domain of CBP is responsible for its neuroprotective function. These studies may have significant bearing on our understanding of genetic basis of AD.

Domain Specific Genetic Mosaic System in the Drosophila Eye

Genetic mosaic approach is commonly used in the Drosophila eye by completely abolishing or misexpressing a gene within a subset of cells to unravel its role during development. Classical genetic mosaic approach involves random clone generation in all developing fields. Consequently, a large sample size needs to be screened to generate and analyze clones in specific domains of the developing eye. To address domain specific functions of genes during axial patterning, we have developed a system for generating mosaic clones by combining Gal4/UAS and flippase (FLP)/FRT system which will allow generation of loss‐of‐function as well as gain‐of‐function clones on the dorsal and ventral eye margins. We used the bifid‐Gal4 driver to drive expression of UAS‐FLP. This reagent can have multiple applications in (i) studying spatio‐temporal function of a gene during dorso‐ventral (DV) axis specification in the eye, (ii) analyzing genetic epistasis of genes involved in DV patterning, and (iii) conducting genome wide screens in a domain specific manner. genesis 51:68–74, 2013.

Molecular Genetic Mechanisms of Axial Patterning: Mechanistic Insights into Generation of Axes in the Developing Eye

All multicellular organisms require axial patterning to transform a single-layer organ primordium to a three-dimensional organ. It involves delineation of anteroposterior (AP), dorsoventral (DV), and proximodistal (PD) axes. Any deviation in this fundamental process results in patterning and growth defects during organogenesis. The Drosophila eye is an excellent model to study axial patterning. In the Drosophila eye, DV lineage is the first axis to be determined, which is followed by generation of the AP axis. The default state of the Drosophila early eye primordium is ventral, and the dorsal fate is established by onset of expression of dorsal eye fate selector pannier (pnr)in a group of cells on the dorsal eye margin. The boundary between dorsal and ventral compartments is the site for activation of Notch (N) signaling and is referred to as the equator. Activation of N signaling is crucial for initiating the cell proliferation and differentiation in the developing Drosophila eye imaginal disc. This chapter will focus on (a) how axial patterning occurs in the developing Drosophila eye, (b) how the developing eye field gets divided into dorsal and ventral cell populations, and (c) how DV patterning genes contribute toward the growth and patterning of the fly retina.

A vertex specific dorsal selector Dve represses the ventral appendage identity in Drosophila head.

Developmental fields are subdivided into lineage-restricted cell populations, known as compartments. In the eye imaginal disc of Drosophila, dorso-ventral (DV) lineage restriction is the primary event, whereas antero-posterior compartment boundary is the first lineage restriction in other imaginal discs. The Iroquois complex (Iro-C) genes function as dorsal selectors and repress the default, ventral, identity in the eye-head primordium. In Iro-C mutant clones, change of the dorsal identity to default ventral fate leads to generation of ectopic DV boundary, which results in dorsal eye enlargement, and duplication of ventral appendages like antenna and maxillary palp. Similar phenotypes were observed in heads with defective proventriculus (dve) mutant clones. Here, we show that the homeobox gene dve is a downstream effector of Iro-C in the dorsal head capsule (vertex) specification and represses the ventral (antennal) identity. Two homeodomain proteins Distal-less (Dll) and Homothorax (Hth) are known to be determinants of the antennal identity. Ectopic antenna formation in heads with dve mutant clones was associated with ectopic Dll expression and endogenous Hth expression in the vertex region. Interestingly, dve Dll double mutant clones could also induce ectopic antennae lacking the distal structures, suggesting that the Dve activity is crucial for repressing inappropriate antenna-forming potential in the vertex region. Our results clearly indicate that not only the activation of effector genes to execute developmental program but also the repression of inappropriate program is crucial for establishment of the organ identity.