Nazo, the Drosophila homolog of the NBIA-mutated protein-c19orf12, is required for triglyceride homeostasis.

Lipid dyshomeostasis has been implicated in a variety of diseases ranging from obesity to neurodegenerative disorders such as Neurodegeneration with Brain Iron Accumulation (NBIA). Here, we uncover the physiological role of Nazo, the Drosophila melanogaster homolog of the NBIA-mutated protein-c19orf12, whose function has been elusive. Ablation of Drosophila c19orf12 homologs leads to dysregulation of multiple lipid metabolism genes. nazo mutants exhibit markedly reduced gut lipid droplet and whole-body triglyceride contents. Consequently, they are sensitive to starvation and oxidative stress. Nazo is required for maintaining normal levels of Perilipin-2, an inhibitor of the lipase-Brummer. Concurrent knockdown of Brummer or overexpression of Perilipin-2 rescues the nazo phenotype, suggesting that this defect, at least in part, may arise from diminished Perilipin-2 on lipid droplets leading to aberrant Brummer-mediated lipolysis. Our findings potentially provide novel insights into the role of c19orf12 as a possible link between lipid dyshomeostasis and neurodegeneration, particularly in the context of NBIA.

Error codes at autopsy to study potential biases in diagnostic error.

OBJECTIVES: Current autopsy practice guidelines do not provide a mechanism to identify potential causes of diagnostic error (DE). We used our autopsy data registry to ask if gender or race were related to the frequency of diagnostic error found at autopsy. METHODS: Our autopsy reports include International Classification of Diseases (ICD) 9 or ICD 10 diagnostic codes for major diagnoses as well as codes that identify types of error. From 2012 to mid-2015 only 2 codes were used: UNDOC (major undocumented diagnoses) and UNCON (major unconfirmed diagnoses). Major diagnoses contributed to death or would have been treated if known. Since mid-2015, codes included specific diagnoses, i.e. undiagnosed or unconfirmed myocardial infarction, infection, pulmonary thromboembolism, malignancy, or other diagnosis as well as cause of death. Adult autopsy cases from 2012 to 2019 were assessed for DE associated with reported sex or race (nonwhite or white). 528 cases were evaluated between 2012 and 2015 and 699 between 2015 and 2019. RESULTS: Major DEs were identified at autopsy in 65.9 % of cases from 2012 to 2015 and in 72.1 % from 2015 to 2019. From 2012 to 2015, female autopsy cases showed a greater frequency in 4 parameters of DE, i.e., in the total number of cases with any error (p=0.0001), in the number of cases with UNDOC errors (p=0.0038) or UNCON errors (p=0.0006), and in the relative proportions of total numbers of errors (p=0.0001). From 2015 to 2019 undocumented malignancy was greater among males (p=0.0065); no other sex-related error was identified. In the same period some DE parameters were greater among nonwhite than among white subjects, including unconfirmed cause of death (p=0.035), and proportion of total error diagnoses (p=0.0003), UNCON diagnoses (p=0.0093), and UNDOC diagnoses (p=0.035). CONCLUSIONS: Coding for DE at autopsy can identify potential effects of biases on diagnostic error.

Application of the condensed protocol for the NIA-AA guidelines for the neuropathological assessment of Alzheimer's disease in an academic clinical practice.

AIMS: In response to concerns regarding resource expenditures required to implement fully the 2012 National Institute on Aging and the Alzheimer's Association (NIA-AA) Sponsored Guidelines for the neuropathological assessment of Alzheimer's disease (AD), we previously developed a sensitive and cost-reducing condensed protocol (CP) at the University of Washington (UW) Alzheimer's Disease Research Center (ADRC) that consolidated the recommended NIA-AA protocol into fewer cassettes requiring fewer immunohistochemical stains. The CP was not designed to replace NIA-AA protocols, but instead to make the NIA-AA criteria accessible to clinical and forensic neuropathology practices where resources limit full implementation of NIA-AA guidelines. METHODS AND RESULTS: In this regard, we developed practical criteria to instigate CP sampling and immunostaining, and applied these criteria in an academic clinical neuropathological practice. During the course of 1 year, 73 cases were sampled using the CP; of those, 53 (72.6%) contained histological features that prompted CP work-up. We found that the CP resulted in increased identification of AD and Lewy body disease neuropathological changes from what was expected using a clinical history-driven work-up alone, while saving approximately $900 per case. CONCLUSIONS: This study demonstrates the feasibility and cost-savings of the CP applied to a clinical autopsy practice, and highlights potentially unrecognised neurodegenerative disease processes in the general ageing community.

FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS.

WIDE AWAKE mediates the circadian timing of sleep onset.

How the circadian clock regulates the timing of sleep is poorly understood. Here, we identify a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of WAKE in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. WAKE levels cycle, peaking near dusk, and the expression of WAKE in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring WAKE expression in LNvs. WAKE interacts with the GABAA receptor Resistant to Dieldrin (RDL), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. We propose that WAKE acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep.

Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila.

Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. Here, we demonstrate that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. We investigate the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function.

Semaphorin regulation of cellular morphology.

Semaphorin proteins, although initially characterized as repulsive neuronal guidance cues, are now appreciated as major contributors to morphogenesis and homeostasis for a wide range of tissue types. Semaphorin-mediated long- and short-range repulsive, and attractive, guidance has profound influences on cellular morphology. The diversity of semaphorin receptor complexes utilized by various semaphorin ligands, the ability of semaphorins themselves to serve as receptors, and the myriad of intracellular signaling components that comprise semaphorin signaling cascades all contribute to cell-type-specific responses to semaphorins. Analysis of the molecular and cellular mechanisms underlying semaphorin function in neural and vascular systems provides insight into principles governing how this large protein family contributes to organogenesis, function, and disease.

Descrambling Dscam diversity.

Neuronal processes exhibit exquisitely complex branching patterns crucial for the formation of distinct neural circuits. In this issue of Cell, Chen et al. (2006) show that the isoform diversity of the Dscam protein in Drosophila is required to establish stereotypical axonal branching patterns, suggesting that nonrandom expression of Dscam alternative splice variants determines neural connectivity.

The spindle checkpoint, aneuploidy, and cancer.

Cancer cells contain abnormal number of chromosomes (aneuploidy), which is a prevalent form of genetic instability in human cancers. Defects in a cell cycle surveillance mechanism called the spindle checkpoint contribute to chromosome instability and aneuploidy. In response to straying chromosomes in mitosis, the spindle checkpoint inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome (APC/C), thus preventing precocious chromosome segregation and ensuring the accurate partition of the genetic material. We review recent progress toward the understanding of the molecular mechanism of the spindle checkpoint and its role in guarding genome integrity at the chromosome level.

Identification of two novel components of the human NDC80 kinetochore complex.

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.