A Quantitative Model of Sporadic Axonal Degeneration in the Drosophila Visual System.

In human neurodegenerative diseases, neurons undergo axonal degeneration months to years before they die. Here, we developed a system modeling early degenerative events in Drosophila adult photoreceptor cells. Thanks to the stereotypy of their axonal projections, this system delivers quantitative data on sporadic and progressive axonal degeneration of photoreceptor cells. Using this method, we show that exposure of adult female flies to a constant light stimulation for several days overcomes the intrinsic resilience of R7 photoreceptors and leads to progressive axonal degeneration. This was not associated with apoptosis. We furthermore provide evidence that loss of synaptic integrity between R7 and a postsynaptic partner preceded axonal degeneration, thus recapitulating features of human neurodegenerative diseases. Finally, our experiments uncovered a role of postsynaptic partners of R7 to initiate degeneration, suggesting that postsynaptic cells signal back to the photoreceptor to maintain axonal structure. This model can be used to dissect cellular and circuit mechanisms involved in the early events of axonal degeneration, allowing for a better understanding of how neurons cope with stress and lose their resilience capacities.SIGNIFICANCE STATEMENT Neurons can be active and functional for several years. In the course of aging and in disease conditions leading to neurodegeneration, subsets of neurons lose their resilience and start dying. What initiates this turning point at the cellular level is not clear. Here, we developed a model allowing to systematically describe this phase. The loss of synapses and axons represents an early and functionally relevant event toward degeneration. Using the ordered distribution of Drosophila photoreceptor axon terminals, we assembled a system to study sporadic initiation of axon loss and delineated a role for non-cell-autonomous activity regulation in the initiation of axon degeneration. This work will help shed light on key steps in the etiology of nonfamilial cases of neurodegenerative diseases.

Cell-Autonomous Control of Neuronal Dendrite Expansion via the Fatty Acid Synthesis Regulator SREBP.

During differentiation, neurons require a high lipid supply for membrane formation as they elaborate complex dendritic morphologies. While glia-derived lipids support neuronal growth during development, the importance of cell-autonomous lipid production for dendrite formation has been unclear. Using Drosophila larva dendritic arborization (da) neurons, we show that dendrite expansion relies on cell-autonomous fatty acid production. The nociceptive class four (CIV) da neurons form particularly large space-filling dendrites. We show that dendrite formation in these CIVda neurons additionally requires functional sterol regulatory element binding protein (SREBP), a crucial regulator of fatty acid production. The dendrite simplification in srebp mutant CIVda neurons is accompanied by hypersensitivity of srebp mutant larvae to noxious stimuli. Taken together, our work reveals that cell-autonomous fatty acid production is required for proper dendritic development and establishes the role of SREBP in complex neurons for dendrite elaboration and function.

The gap junction protein Innexin3 is required for eye disc growth in Drosophila.

The Drosophila compound eye develops from a bilayered epithelial sac composed of an upper peripodial epithelium layer and a lower disc proper, the latter giving rise to the eye itself. During larval stages, complex signalling events between the layers contribute to the control of cell proliferation and differentiation in the disc. Previous work in our lab established the gap junction protein Innexin2 (Inx2) as crucial for early larval eye disc growth. By analysing the contribution of other Innexins to eye size control, we have identified Innexin3 (Inx3) as an important growth regulator. Depleting inx3 during larval eye development reduces eye size, while elevating inx3 levels increases eye size, thus phenocopying the inx2 loss- and gain-of-function situation. As demonstrated previously for inx2, inx3 regulates disc cell proliferation and interacts genetically with the Dpp pathway, being required for the proper activation of the Dpp pathway transducer Mad at the furrow and the expression of Dpp receptor Punt in the eye disc. At the developmental timepoint corresponding to eye disc growth, Inx3 colocalises with Inx2 in disc proper and peripodial epithelium cell membranes. In addition, we show that Inx3 protein levels critically depend on inx2 throughout eye development and that inx3 modulates Inx2 protein levels in the larval eye disc. Rescue experiments demonstrate that Inx3 and Inx2 cooperate functionally to enable eye disc growth in Drosophila. Finally, we demonstrate that expression of Inx3 and Inx2 is not only needed in the disc proper but also in the peripodial epithelium to regulate growth of the eye disc. Our data provide a functional demonstration that putative Inx2/Inx3 heteromeric channels regulate organ size.

Drosophila eye size is determined by Innexin 2-dependent Decapentaplegic signalling.

Organogenesis relies on specific genetic and molecular programmes, which orchestrate growth and cellular differentiation over developmental time. This is particularly important during Drosophila eye development in which cell-cell inductive events and long-range signalling have to be integrated to regulate proper cell proliferation, differentiation and morphogenesis. How these processes are coordinated is still not very well understood. Here we identify the gap junction protein Innexin2 (Inx2) as an important regulator of eye development. Depleting inx2 during eye development reduces eye size whereas elevating inx2 levels increases eye size. Loss- and gain-of-function experiments demonstrate that inx2 is required functionally in larval eye disc cells where it localises apico-laterally. inx2 regulates disc cell proliferation as well as morphogenetic furrow movement and as a result the amount of differentiated photoreceptors. inx2 interacts genetically with the Dpp pathway and we find that proper activation of the Dpp pathway transducer Mad at the furrow and expression of Dpp receptors Thickveins and Punt in the anterior disc compartment require inx2. We further show that inx2 is required for the transcriptional activation of dpp and punt in the eye disc. Our results highlight the crucial role of gap junction proteins in regulating morphogen-dependent organ size determination.

Extracellular phosphorylation of the amyloid ß-peptide promotes formation of toxic aggregates during the pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid ß-peptides (Aß) in the brain. Aß derives by sequential proteolytic processing of the amyloid precursor protein by ß- and γ-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aß domain promote the formation of neurotoxic Aß assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aß and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aß undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aß assemblies that represent nuclei for fibrillization. Phosphorylated Aß was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aß. Phosphorylation of Aß could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD.

A role for the extracellular domain of Crumbs in morphogenesis of Drosophila photoreceptor cells.

Morphogenesis of Drosophila photoreceptor cells includes the subdivision of the apical membrane into the photosensitive rhabdomere and the associated stalk membrane, as well as a considerable elongation of the cell. Drosophila Crumbs (Crb), an evolutionarily conserved transmembrane protein, organizes an apical protein scaffold, which is required for elongation of the photoreceptor cell and extension of the stalk membrane. To further elucidate the role played by different Crb domains during eye morphogenesis, we performed a structure-function analysis in the eye. The analysis showed that the three variants tested, namely full-length Crb, the membrane-bound intracellular domain and the extracellular domain were able to rescue the elongation defects of crb mutant rhabdomeres. However, only full-length Crb and the membrane-bound intracellular domain could partially restore the length of the stalk membrane, while the extracellular domain failed to do so. This failure was associated with the inability of the extracellular domain to recruit beta(Heavy)-spectrin to the stalk membrane. These results highlight the functional importance of the extracellular domain of Crb in the Drosophila eye. They are in line with previous observations, which showed that mutations in the extracellular domain of human CRB1 are associated with retinitis pigmentosa 12 and Leber congenital amaurosis, two severe forms of retinal dystrophy.

Towards understanding CRUMBS function in retinal dystrophies.

Mutations in the Crumbs homologue 1 (CRB1) gene cause autosomal recessive retinitis pigmentosa (arRP) and autosomal Leber congenital amaurosis (arLCA). The crumbs (crb) gene was originally identified in Drosophila and encodes a large transmembrane protein required for maintenance of apico-basal cell polarity and adherens junction in embryonic epithelia. Human CRB1 and its two paralogues, CRB2 and CRB3, are highly conserved throughout the animal kingdom. Both in Drosophila and in vertebrates, the short intracellular domain of Crb/CRB organizes an evolutionary conserved protein scaffold. Several lines of evidence, obtained both in Drosophila and in mouse, show that loss-of-function of crb/CRB1 or some of its intracellular interactors lead to morphological defects and light-induced degeneration of photoreceptor cells, features comparable to those observed in patients lacking CRB1 function. In this review, we describe how understanding Crb complex function in fly and vertebrate retina enhances our knowledge of basic cell biological processes and might lead to new therapeutic approaches for patients affected with retinal dystrophies caused by mutations in the CRB1 gene.

DPATJ plays a role in retinal morphogenesis and protects against light-dependent degeneration of photoreceptor cells in the Drosophila eye.

The establishment of apicobasal polarity in epithelial cells is a prerequisite for their function. Drosophila photoreceptor cells derive from epithelial cells, and their apical membranes undergo elaborate differentiation during pupal development, forming photosensitive rhabdomeres and associated stalk membranes. Crumbs (Crb), a transmembrane protein involved in the maintenance of epithelial polarity in the embryo, defines the stalk as a subdomain of the apical membrane. Crb organizes a complex composed of several PDZ domain-containing proteins, including DPATJ (formerly known as Discs lost). Taking advantage of a DPATJ mutant line in which only a truncated form of the protein is synthesized, we demonstrate that DPATJ is necessary for the stability of the Crb complex at the stalk membrane and is crucial for stalk membrane development and rhabdomere maintenance during late pupal stages. Moreover, DPATJ protects against light-induced photoreceptor degeneration.

Interleukin-9 promotes eosinophilic rejection of mouse heart allografts.

BACKGROUND: Eosinophils participate in allograft rejection when donor-reactive helper T lymphocytes are T-helper type 2 (Th2)-biased. Whereas the involvement of interleukin (IL)-4 and IL-5 in these forms of rejection is well established, the role of IL-9, another Th2-type cytokine promoting eosinophilia, has not been determined. METHODS: We first used real-time polymerase chain reaction to quantify IL-9 mRNA in rejected allografts in a mouse model of fully mismatched heart transplantation in which recipients were devoid of CD8 T cells and developed a Th2 alloimmune response. We then compared allograft survival in wild-type versus IL-9-deficient mice depleted of CD8 T cells. Finally, we compared the fate of major histocompatibility complex class II-mismatched cardiac transplants from wild-type versus IL-9 transgenic donors to determine the influence of IL-9 overexpression within the graft. RESULTS: The Th2 alloimmune response in CD8-deficient mice was associated with the accumulation of IL-9 mRNA in the rejected graft. In IL-9-deficient recipients depleted of CD8 T cells, eosinophil infiltration of heart allografts did not develop, but rejection still occurred. In the major histocompatibility complex class II disparate model, heart allografts from IL-9 transgenic donors were acutely rejected, whereas grafts from wild-type donors did not develop rejection. Acute rejection of IL-9 transgenic hearts was associated with massive eosinophil infiltration and prevented by neutralization of either IL-4 or IL-5. CONCLUSION: IL-9 is critically involved in heart transplant eosinophilia in conjunction with IL-4 and IL-5.