Physiological significance of WDR45, a responsible gene for β-propeller protein associated neurodegeneration (BPAN), in brain development

WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause β-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay with intellectual disability, the neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in excitatory synapses by biochemical fractionation. Since WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by in utero electroporation in mice to recapitulate the pathophysiological conditions of BPAN. Knockdown of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.

Exploiting flow cytometry for the unbiased quantification of protein inclusions in Caenorhabditis elegans

The aggregation of proteins into inclusions or plaques is a prominent hallmark of a diverse range of pathologies including neurodegenerative diseases. The quantification of such inclusions in Caenorhabditis elegans models of aggregation is usually achieved by fluorescence microscopy or other techniques involving biochemical fractionation of worm lysates. Here, we describe a simple and rapid flow cytometry-based approach that allows fluorescently-tagged inclusions to be enumerated in whole worm lysate in a quantitative and unbiased fashion. We demonstrate that this technique is applicable to multiple C. elegans models of aggregation and importantly, can be used to monitor the dynamics of inclusion formation in response to heat shock and during aging. This includes the characterisation of physicochemical properties of inclusions, such as their size, which may reveal how aggregate formation is distinct in different tissues or at different stages of pathology or aging. This new method can be used as a powerful technique for the medium- to high-throughput quantification of inclusions in future studies of genetic or chemical modulators of aggregation in C. elegans.

UbiB proteins regulate cellular CoQ distribution in Saccharomyces cerevisiae

Beyond its role in mitochondrial bioenergetics, Coenzyme Q (CoQ, ubiquinone) serves as a key membrane-embedded antioxidant throughout the cell. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of Saccharomyces cerevisiae genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally affect this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids, whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8—an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal protein machinery central to CoQ trafficking in yeast and lend insights into the broader interplay between mitochondria and the rest of the cell.

Mitochondrial LonP1 protease is implicated in the degradation of unstable Parkinson's disease-associated DJ-1/PARK 7 missense mutants

DJ-1/PARK7 mutations are linked with familial forms of early-onset Parkinson's disease (PD). We have studied the degradation of untagged DJ-1 wild type (WT) and missense mutants in mouse embryonic fibroblasts obtained from DJ-1-null mice, an approach closer to the situation in patients carrying homozygous mutations. The results showed that the mutants L10P, M26I, A107P, P158Δ, L166P, E163K, and L172Q are unstable proteins, while A39S, E64D, R98Q, A104T, D149A, A171S, K175E, and A179T are as stable as DJ-1 WT. Inhibition of proteasomal and autophagic-lysosomal pathways had little effect on their degradation. Immunofluorescence and biochemical fractionation studies indicated that M26I, A107P, P158Δ, L166P, E163K, and L172Q mutants associate with mitochondria. Silencing of mitochondrial matrix protease LonP1 produced a strong reduction of the degradation of the mitochondrial-associated DJ-1 mutants A107P, P158Δ, L166P, E163K, and L172Q but not of mutant L10P. These results demonstrated a mitochondrial pathway of degradation of those DJ-1 missense mutants implicated in PD pathogenesis.

Physiological Significance of WDR45, A Responsible Gene for β-propeller Protein Associated Neurodegeneration (BPAN), in Brain Development

WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause b-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay including intellectual disability, neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in the excitatory synapses in biochemical fractionation. Since the WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by an in utero electroporation method with mice to recapitulate the pathophysiological conditions of BPAN. Silencing of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to the control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.

UbiB proteins regulate cellular CoQ distribution

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid essential for many core metabolic processes in mitochondria, including oxidative phosphorylation1-3. While lesser appreciated, CoQ also serves as a key membrane-embedded antioxidant throughout the cell4. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of yeast genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally regulate this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids (PUFAs), whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8—an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal new protein machinery central to CoQ trafficking in yeast and lend new insights into the broader interplay between mitochondrial and cellular processes.

Proximity-based proteomics reveals the thylakoid lumen proteome in the cyanobacterium Synechococcus sp. PCC 7002

Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.

No evidence for Ago2 translocation from the host erythrocyte into the Plasmodium parasite

Background: Plasmodium parasites rely on various host factors to grow and replicate within red blood cells (RBC). While many host proteins are known that mediate parasite adhesion and invasion, few examples of host enzymes co-opted by the parasite during intracellular development have been described. Recent studies suggested that the host protein Argonaute 2 (Ago2), which is involved in RNA interference, can translocate into the parasite and affect its development. Here, we investigated this hypothesis. Methods: We used several different monoclonal antibodies to test for Ago2 localisation in the human malaria parasite, P. falciparum and rodent P. berghei parasites. In addition, we biochemically fractionated infected red blood cells to localize Ago2. We also quantified parasite growth and sexual commitment in the presence of the Ago2 inhibitor BCI-137. Results: Ago2 localization by fluorescence microscopy produced inconclusive results across the three different antibodies, suggesting cross-reactivity with parasite targets. Biochemical separation of parasite and RBC cytoplasm detected Ago2 only in the RBC cytoplasm and not in the parasite. Inhibition of Ago2 using BCl-137 did not result in altered parasite development. Conclusion: Ago2 localization in infected RBCs by microscopy is confounded by non-specific binding of antibodies. Complementary results using biochemical fractionation and Ago2 detection by western blot did not detect the protein in the parasite cytosol, and growth assays using a specific inhibitor demonstrated that its catalytical activity is not required for parasite development. We therefore conclude that previous data localising Ago2 to parasite ring stages are due to antibody cross reactivity, and that Ago2 is not required for intracellular Plasmodium development.

Fer1L5, a Dysferlin Homologue Present in Vesicles and Involved in C2C12 Myoblast Fusion and Membrane Repair

Fer1L5 is a dysferlin and myoferlin related protein, which has been predicted to have a role in vesicle trafficking and muscle membrane fusion events. Mutations in dysferlin and otoferlin genes cause heredity diseases: muscular dystrophy and deafness in humans, respectively. Dysferlin is implicated in membrane repair. Myoferlin has a role in myogenesis. In this study, we investigated the role of the Fer1L5 protein during myoblast fusion and membrane repair. To study the functions of Fer1L5 we used confocal microscopy, biochemical fractionation, Western blot analysis and multiphoton laser wounding assay. By immunolabelling, Fer1L5 was detected in vesicular structures. By biochemical fractionation Fer1L5 was observed in low density vesicles. Our studies show that the membranes of Fer1L5 vesicles are non-resistant to non-ionic detergent. Partial co-staining of Fer1L5 with other two ferlin vesicles, respectively, was observed. Fer1L5 expression was highly detected at the fusion sites of two apposed C2C12 myoblast membranes and its expression level gradually increased at D2 and reached a maximum at day 4 before decreasing during further differentiation. Our studies showed that Fer1L5 has fusion defects during myoblast fusion and impaired membrane repair when the C2C12 cultures were incubated with inhibitory Fer1L5 antibodies. In C2C12 cells Fer1L5 vesicles are involved in two stages, the fusion of myoblasts and the formation of large myotubes. Fer1L5 also plays a role in membrane repair.