Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.

Physiological and pathological roles of FATP-mediated lipid droplets in Drosophila and mice retina.

Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer's and Parkinson's diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types in response to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient for expansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic and promoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-metFB Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions.

p53-dependent programmed necrosis controls germ cell homeostasis during spermatogenesis.

The importance of regulated necrosis in pathologies such as cerebral stroke and myocardial infarction is now fully recognized. However, the physiological relevance of regulated necrosis remains unclear. Here, we report a conserved role for p53 in regulating necrosis in Drosophila and mammalian spermatogenesis. We found that Drosophila p53 is required for the programmed necrosis that occurs spontaneously in mitotic germ cells during spermatogenesis. This form of necrosis involved an atypical function of the initiator caspase Dronc/Caspase 9, independent of its catalytic activity. Prevention of p53-dependent necrosis resulted in testicular hyperplasia, which was reversed by restoring necrosis in spermatogonia. In mouse testes, p53 was required for heat-induced germ cell necrosis, indicating that regulation of necrosis is a primordial function of p53 conserved from invertebrates to vertebrates. Drosophila and mouse spermatogenesis will thus be useful models to identify inducers of necrosis to treat cancers that are refractory to apoptosis.

Spen is required for pigment cell survival during pupal development in Drosophila.

Apoptosis is required during development to eliminate superfluous cells and sculpt tissues; spatial and timed control of apoptosis ensures that the necessary number of cells is eliminated at a precise time in a given tissue. The elimination of supernumerary pigment or inter-ommatidial cells (IOCs) depends on cell-cell communication and is necessary for the formation of the honeycomb-like structure of the Drosophila eye. However, the mechanisms occurring during pupal development and controlling apoptosis of superfluous IOC in space and time remain unclear. Here, we found that split-ends (spen) is required for IOC survival at the time of removal of superfluous IOCs. Loss of spen function leads to abnormal removal of IOCs by apoptosis. We show that spen is required non-autonomously in cone cells for the survival of IOCs by positively regulating the Spitz/EGFR pathway. We propose that Spen is an important survival factor that ensures spatial control of the apoptotic wave that is necessary for the correct patterning and formation of the Drosophila eye.

Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival.

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.

ER stress protects from retinal degeneration.

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.

OTX5 regulates pineal expression of the zebrafish REV-ERB alpha through a new DNA binding site.

The pineal gland plays a central role in the photoneuroendocrine system and acts as a photosensory organ in lower vertebrates. The orphan nuclear receptor Rev-erbalpha (NR1D1) has previously been shown to be expressed in the pineal and to be regulated with a robust circadian rhythm during zebrafish embryogenesis. This early pineal expression is under the control of the transcription factor Orthodenticle homeobox 5 (Otx5). In this paper, we show that Otx5 regulates the second zfRev-erbalpha promoter, ZfP2. Despite the absence of a classical Otx-binding site within ZfP2, this regulation depends on the integrity of the Otx5 homeodomain. Mapping experiments as well as EMSAs show that this interaction between Otx5 and ZfP2 depends on a noncanonical bipartite Otx-binding site (GANNCTTA and TAAA) that we called pineal expression related element (PERE). We showed that PERE is necessary for pineal expression in vivo by injecting zebrafish embryos with wild type and mutated versions of zfRev-erbalpha promoter fused to green fluorescent protein. Interestingly, PERE is found upstream of other genes expressed in the pineal gland, suggesting that it may play an important role in governing pineal expression. Our data establish that PERE is a novel cis-acting element contributing to pineal-specific gene expression and to Otx target gene regulation.

Drosophila p53 integrates the antagonism between autophagy and apoptosis in response to stress.

The tumor suppressor TP53/p53 is a known regulator of apoptosis and macroautophagy/autophagy. However, the molecular mechanism by which TP53 regulates 2 apparently incompatible processes remains unknown. We found that Drosophila lacking p53 displayed impaired autophagic flux, higher caspase activation and mortality in response to oxidative stress compared with wild-type flies. Moreover, autophagy and apoptosis were differentially regulated by the p53 (p53B) and ΔNp53 (p53A) isoforms: while the former induced autophagy in differentiated neurons, which protected against cell death, the latter inhibited autophagy by activating the caspases Dronc, Drice, and Dcp-1. Our results demonstrate that the differential use of p53 isoforms combined with the antagonism between apoptosis and autophagy ensures the generation of an appropriate p53 biological response to stress.

A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors.

BACKGROUND: Dynamic monitoring of protein expression and localization is fundamental to the understanding of biological processes. The paired-class homeodomain-containing transcription factor Otx2 is essential for normal head and brain development in vertebrates. Recent conditional knockout studies have pointed to multiple roles of this protein during late development and post-natal life. Yet, later expression and functions remain poorly characterized as specific reagents to detect the protein at any stage of development are still missing. RESULTS: We generated a new mouse line harbouring an insertion of the GFP gene within the Otx2 coding sequence to monitor the gene activity while preserving most of its functions. Our results demonstrate that this line represents a convenient tool to capture the dynamics of Otx2 gene expression from early embryonic stages to adulthood. In addition, we could visualize the intracellular location of Otx2 protein. In the retina, we reinterpret the former view of protein distribution and show a further level of regulation of intranuclear protein localization, which depends on the cell type. CONCLUSION: The GFP-tagged Otx2 mouse line fully recapitulates previously known expression patterns and brings additional accuracy and easiness of detection of Otx2 gene activity. This opens up the way to live imaging of a highly dynamic actor of brain development and can be adapted to any mutant background to probe for genetic interaction between Otx2 and the mutated gene.

Molecular dissection reveals decreased activity and not dominant negative effect in human OTX2 mutants.

The paired-type homeodomain transcription factor Otx2 is essential for forebrain and eye development. Severe ocular malformations in humans have recently been associated with heterozygous OTX2 mutations. To document the molecular defects in human mutants, Otx2 structural characterization was carried out. A collection of deletion and point mutants was created to perform transactivation, DNA binding, and subcellular localization analyses. Transactivation was ascribed to both N- and C-termini of the protein, and DNA binding to the minimal homeodomain, where critical amino acid residues were identified. Acute nuclear localization appeared controlled by a nuclear localization sequence located within the homeodomain which acts in conjunction with a novel nuclear retention domain that we unraveled located in the central part of the protein. This region, which is poorly conserved among Otx proteins, was also endowed with dominant negative activity suggesting that it might confer unique properties to Otx2. Molecular diagnostic of human mutant OTX2 proteins discriminates hypomorphic and loss of function mutations from other mutations that may not be relevant to ocular pathology.

Temporal and spatial delineation of mouse Otx2 functions by conditional self-knockout.

To identify the independent spatial and temporal activities of the essential developmental gene the Otx2, the germline mutation of which is lethal at embryonic day 8.5, we floxed one allele and substituted the other with an inducible CreER recombinase gene. This makes 'trans' self-knockout possible at any developmental stage. The transient action of tamoxifen pulses allows time-course mutation. We demonstrate efficient temporal knockout and demarcate spatio-temporal windows in which Otx2 controls the head, brain structures and body development.

Alternative usage of Otx2 promoters during mouse development.

Our previous structural analysis of mouse Otx2 transcripts has revealed the existence of three different promoters and suggested that the corresponding mRNAs could exhibit specific expression patterns. Here, we analyze the precise dynamics of their expression throughout mouse development. Their spatial distribution was determined by isoform-specific in situ hybridization and their relative abundance by real-time reverse transcriptase-polymerase chain reaction. Although the three promoters may be used in the same areas, we show that transcription preferentially occurs from the proximal promoter at onset of gene activity in early embryogenesis, and switches to the more distal one in most of the sites of expression in the adult brain. During gestation, their relative utilization becomes inverted. The third promoter, which shows no activity in embryonic stem cells and is moderately expressed during embryogenesis, is mostly used in specific areas derived from the rostral part of the neural tube.

New Otx2 mRNA isoforms expressed in the mouse brain.

The mouse Otx2 gene is essential throughout head and brain development, from anterior-posterior polarity determination and neuroectoderm induction to post-natal sensory organ maturation. These numerous activities must rely on a very finely tuned regulation of expression. In order to understand the molecular control of the Otx2 gene, we set out to isolate its promoter. During this quest, we identified three remote transcription start sites, two defining two new upstream exons and one mapping within the previously reported first exon. The three transcripts differed in their 5' non-coding region but encoded the same protein. The transcription start nucleotides of each mRNA species have been mapped by RNase protection assays and by an RNA circularization technique. We have demonstrated that they are all used and linked to functional promoters. In addition to leader versatility, we also detected alternative splicing within the coding sequence that gives rise to a new protein endowed with an 8 amino-acid insertion upstream of the homeodomain. Combined analysis of the relative abundance of Otx2 mRNA isoforms in representative tissues and in situ hybridization studies revealed distinct spatial and temporal, although partially overlapping, expression patterns of the mRNA isoforms. These findings provide new clues to a better understanding of the relationships between Otx2 gene architecture and its complex regulatory requirements.