BackgroundArtemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates.ResultsIn the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype.ConclusionsThe decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

Afadin 6 (AF-6) is an F-actin binding multidomain-containing scaffolding protein that is known for its function in cell-cell adhesion. Interestingly, besides this well documented role, we recently found that AF-6 is a Parkin-interacting protein that augments Parkin/PINK1-mediated mitophagy. Notably, mutations in Parkin and PINK1 are causative of recessively inherited forms of Parkinson’s disease (PD) and aberrant mitochondrial homeostasis is thought to underlie PD pathogenesis. Given the novel role of AF-6 in mitochondrial quality control (QC), we hypothesized that AF-6 overexpression may be beneficial to PD. Using the Drosophila melanogaster as a model system, we demonstrate in this study that transgenic overexpression of human AF-6 in parkin and also pink1 null flies rescues their mitochondrial pathology and associated locomotion deficit, which results in their improved survival over time. Similarly, AF-6 overexpression also ameliorates the pathological phenotypes in flies expressing the Leucine Rich Repeat Kinase 2 (LRRK2) G2019S mutant, a mutation that is associated with dominantly-inherited PD cases in humans. Conversely, when endogenous AF-6 expression is silenced, it aggravates the disease phenotypes of LRRK2 mutant flies. Aside from these genetic models, we also found that AF-6 overexpression is protective against the loss of dopaminergic neurons in flies treated with rotenone, a mitochondrial complex I inhibitor commonly used to generate animal models of PD. Taken together, our results demonstrate that AF-6 protects against dopaminergic dysfunction and mitochondrial abnormalities in multiple Drosophila models of PD, and suggest the therapeutic value of AF-6-related pathways in mitigating PD pathogenesis.

Drosophila blue cheese ( bchs ) encodes a BEACH domain adaptor protein that, like its human homolog ALFY, promotes clearance of aggregated proteins through its interaction with Atg5 and p62. bchs mutations lead to age-dependent accumulation of ubiquitinated inclusions and progressive neurodegeneration in the fly brain, but neither the influence of autophagy on bchs -related degeneration, nor bchs’ placement in the autophagic hierarchy have been shown. We present epistatic evidence in a well-defined larval motor neuron paradigm that in bchs mutants, synaptic accumulation of ubiquitinated aggregates and neuronal death can be rescued by pharmacologically amplifying autophagic initiation. Further, pharmacological rescue requires at least one intact BEACH-containing isoform of the two identified in this study. Genetically augmenting a late step in autophagy, however, rescues even a strong mutation which retains only a third, non-BEACH containing isoform. Using living primary larval brain neurons, we elucidate the primary defect in bchs to be an excess of early autophagic compartments and a deficit in mature compartments. Conversely, rescuing the mutants by full-length Bchs over-expression induces mature compartment proliferation and rescues neuronal death. Surprisingly, only the longest Bchs isoform colocalizes well with autophagosomes, and shuttles between different vesicular locations depending on the type of autophagic impetus applied. Our results are consistent with Bchs promoting autophagic maturation, and the BEACH domain being required for this function. HIGHLIGHTS The autophagic adaptor blue cheese is placed in an epistatic hierarchy, using pharmacological and genetic modulation of bchs - motor neuron degeneration. An intact BEACH isoform can promote autophagic proliferation, and in primary larval brain neurons Bchs shuttles to different components of the autophagy machinery, dependent on the stimulus.

Mutations in LRRK2 are currently recognized as the most common monogenetic cause of Parkinsonism. The elevation of kinase activity of LRRK2 that frequently accompanies its mutations is widely thought to contribute to its toxicity. Accordingly, many groups have developed LRRK2-specific kinase inhibitors as a potential therapeutic strategy. Given that protein phosphorylation is a reversible event, we sought to elucidate the phosphatase(s) that can reverse LRRK2-mediated phosphorylation, with the view that targeting this phosphatase(s) may similarly be beneficial. Using an unbiased RNAi phosphatase screen conducted in a Drosophila LRRK2 model, we identified PP2A as a genetic modulator of LRRK2-induced neurotoxicity. Further, we also identified ribosomal S6 kinase (S6K), a target of PP2A, as a novel regulator of LRRK2 function. Finally, we showed that modulation of PP2A or S6K activities ameliorates LRRK2-associated disease phenotype in Drosophila.

Neurodegenerative disorders are becoming an increasing concern in today's aging society whereby cognitive, postural and memory abilities are impaired in sporadic and familial forms of the disorders. Extensive research over the years has generated insights into the pathophysiological mechanisms of proteins implicated in the etiology of these diseases. A common pathological hallmark is the accumulation of cytotoxic protein aggregates in the brain that accompanies neuronal dysfunction and atrophy. The intracellular protein quality control system consists of three main components: chaperone-mediated folding, the ubiquitin-proteasome system, and autophagy. In this study, it is shown that autophagy function is impaired in Drosophila melanogaster neurodegenerative mutants in the gene blue cheese (bchs), and conversely that augmenting autophagy is sufficient to rescue the degeneration. We show this by rescuing neuronal atrophy through rapamycin feeding and atg7 over-expression. Rapamycin feeding also alleviates the accumulation of ubiquitinated aggregates in neuronal termini of bchs motor neurons. Autophagy inhibitors wortmannin and 3-methyladenine in contrast exacerbate neuronal death in bchs mutants. In the absence of Bchs protein, AtgS-positive and Atg8-positive autophagic vacuoles are not able to be recruited to ubiquitinated aggregates. Colocalization analysis and live imaging on primary neurons show that Bchs dissociates from Rabll and associates with AtgS during Huntingtin Q93 expression. However, there is no effect on the association of Bchs with Atg8 or Spinster, a late endolysosomal marker, in response to the induction of aggrephagy. These findings suggest that Bchs is involved in the early steps of autophagosome formation and recognition of target substrates, in a similar manner as the human homologue Autophagy Linked FYVE (ALFY) protein. Bchs is unlikely to be a motor protein adaptor mediating cargo assembly due to the absence of interaction with dynein or kinesin compared to APP-Iike interacting protein 1 (Aplipl). Therefore, this study demonstrates that Bchs functions as an adaptor bridging the autophagy machinery to aggregate-prone proteins using a neurodegenerative disease model in Drosophila melanogaster.