Inhibition of Autophagy Alters Intracellular Transport of APP Resulting in Increased APP Processing.

Alzheimer's disease (AD) pathology is characterized by amyloid beta (Aß) plaques and dysfunctional autophagy. Aß is generated by sequential proteolytic cleavage of amyloid precursor protein (APP), and the site of intracellular APP processing is highly debated, which may include autophagosomes. Here, we investigated the involvement of autophagy, including the role of ATG9 in APP intracellular trafficking and processing by applying the RUSH system, which allows studying the transport of fluorescently labeled mCherry-APP-EGFP in a systematic way, starting from the endoplasmic reticulum. HeLa cells, expressing the RUSH mCherry-APP-EGFP system, were investigated by live cell imaging, immunofluorescence, and Western blot. We found that mCherry-APP-EGFP passed through the Golgi faster in ATG9 knockout cells. Furthermore, ATG9 deletion shifted mCherry-APP-EGFP from early endosomes and lysosomes toward the plasma membrane concomitant with reduced endocytosis. Importantly, this alteration in mCherry-APP-EGFP transport resulted in increased secreted mCherry-soluble APP and C-terminal fragment-EGFP. These effects were also phenocopied by pharmacological inhibition of ULK1, indicating that autophagy is regulating the intracellular trafficking and processing of APP. These findings contribute to the understanding of the role of autophagy in APP metabolism and could potentially have implications for new therapeutic approaches for AD.

Amyloid precursor protein elevates fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load.

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates consisting of its adapter protein FE65, the histone acetyltransferase TIP60 and the tumour suppressor proteins p53 and PML. APP C-terminal (APP-CT50) complexes co-localize and co-precipitate with p53 and PML. The PML nuclear body generation is induced and fusion occurs over time depending on APP signalling and STED imaging revealed active gene expression within the complex. We further show that the nuclear aggregates of APP-CT50 fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimer's disease brains reveal a highly significant reduction of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT50 signalling to the nucleus takes place in the aged human brain and is involved in the pathophysiology of AD.

Stage-specific action of matrix metalloproteinases influences progressive hereditary kidney disease.

BACKGROUND: Glomerular basement membrane (GBM), a key component of the blood-filtration apparatus in the in the kidney, is formed through assembly of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations or deletions involving alpha3(IV), alpha4(IV), or alpha5(IV) chains of type IV collagen in the GBM have been identified as the cause for Alport syndrome in humans, a progressive hereditary kidney disease associated with deafness. The pathological mechanisms by which such mutations lead to eventual kidney failure are not completely understood. METHODS AND FINDINGS: We showed that increased susceptibility of defective human Alport GBM to proteolytic degradation is mediated by three different matrix metalloproteinases (MMPs)--MMP-2, MMP-3, and MMP-9--which influence the progression of renal dysfunction in alpha3(IV)-/- mice, a model for human Alport syndrome. Genetic ablation of either MMP-2 or MMP-9, or both MMP-2 and MMP-9, led to compensatory up-regulation of other MMPs in the kidney glomerulus. Pharmacological ablation of enzymatic activity associated with multiple GBM-degrading MMPs, before the onset of proteinuria or GBM structural defects in the alpha3(IV)-/- mice, led to significant attenuation in disease progression associated with delayed proteinuria and marked extension in survival. In contrast, inhibition of MMPs after induction of proteinuria led to acceleration of disease associated with extensive interstitial fibrosis and early death of alpha3(IV)-/- mice. CONCLUSIONS: These results suggest that preserving GBM/extracellular matrix integrity before the onset of proteinuria leads to significant disease protection, but if this window of opportunity is lost, MMP-inhibition at the later stages of Alport disease leads to accelerated glomerular and interstitial fibrosis. Our findings identify a crucial dual role for MMPs in the progression of Alport disease in alpha3(IV)-/- mice, with an early pathogenic function and a later protective action. Hence, we propose possible use of MMP-inhibitors as disease-preventive drugs for patients with Alport syndrome with identified genetic defects, before the onset of proteinuria.

Use of micro-XANES to speciate chromium in airborne fine particles in the Sacramento Valley.

While particulate matter (PM) in the atmosphere can lead to a wide array of negative health effects, the cause of toxicity is largely unknown. One aspect of PM that likely affects health is the chemical composition, in particular the transition metals within the particles. Chromium is one transition metal of interest due to its two major oxidation states, with Cr(III) being much less toxic compared to Cr(VI). Using microfocused X-ray absorption near edge structure (micro-XANES), we analyzed the Cr speciation in fine particles (diameters < or = 2.5 microm) collected at three sites in the Sacramento Valley of northern California. The microfocused X-ray beam enables us to look at very small areas on the filter with a resolution of typically 5-7 micrometers. With XANES we are able to not only distinguish between Cr(VI) and Cr(III), but also to identify different types of Cr(III) and more reduced Cr species. At all of our sampling sites the main Cr species were Cr(III), with Cr(OH)3 or a Cr-Fe, chromite-like, phase being the dominant species. Cr(VI)-containing particles were found only in the most urban site. All three sites contained some reduced Cr species, either Cr(0) or Cr3C2, although these were minor components. This work demonstrates that micro-XANES can be used as a minimally invasive analytical tool to investigate the composition of ambient PM.

Comparison of N,N'-diarylsquaramides and N,N'-diarylureas as antagonists of the CXCR2 chemokine receptor.

N,N'-diarylsquaramides were prepared and evaluated as antagonists of CXCR2. The compounds were found to be potent and selective antagonists of CXCR2. Significant differences in SAR was observed relative to the previously described N,N'-diarylurea series. As was the case in the N,N'-diarylurea series, placing sulfonamide substituent adjacent to the acidic phenol significantly reduced the clearance in rat pharmacokinetic studies.

A comparison of household lead exposure assessment methods in an old, urban community.

The accurate assessment of lead hazards within the household is critical to the prevention of lead exposure to children living in urban communities. Although dust wipes are currently accepted as the best method of assessing lead hazards, questionnaires and visual inspections are also used. This study evaluates the level of agreement among these three methods of assessment using a sample of 126 women living in old, urban houses. The level of agreement was assessed using the kappa statistic, which adjusts for chance agreement. Overall, the kappa results for both the participant's assessment (questionnaire) and the visual inspector's assessment were low, indicating only slight to moderate agreement with the dust lead wipe levels. Kappas were higher and more consistent for the visual inspector's assessment than for the participant's assessment. These results indicate that visual inspections and participant questionnaires may provide less accurate information regarding lead hazards within the household than dust wipes. Because lead hazard recognition is important in the prevention of lead exposure in children, our data suggest that emphasis should be placed on the measurement of dust lead levels directly in assessing household lead hazards, possibly by teaching women in high-risk neighborhoods to take dust samples themselves.

Recruitment of older women: lessons learned from the Baltimore Hip Studies.

OBJECTIVES: This study used a qualitative approach in which participants were asked to write about their experiences in recruiting older women into either one of two exercise intervention studies that are part of the Baltimore Hip Studies. The sample included 8 researcher nurses all women, White, and 42-53 years of age. BACKGROUND: Older adults, particularly older women, are less likely to participate in research studies when compared to their younger counterparts. The purpose of this study was to explore the techniques successfully used by research nurses in the Baltimore Hip Studies to recruit older women after hip fracture into exercise intervention studies. METHOD: Data analysis was performed using basic content analysis (Crabtree & Miller, 1992; Miles & Huberman, 1984) "in vivo" coding (Dowd, 1991), or "grounded" coding (Glaser & Strauss, 1967), which involves using the informants' own words to capture a particular idea. RESULTS: A total of 16 codes were identified and reduced to nine themes. Seven themes focused on techniques that facilitated recruitment: (a) caring for individuals; (b) emphasizing benefits; (c) eliciting support from others; (d) being an expert; (e) using role models; (f) using good timing; and (g) giving good first impressions. The remaining two themes identified barriers to recruitment: (a) time commitment and (b) lack of support. DISCUSSION: Based on these themes, specific recruitment techniques are recommended. Ongoing research, however, is needed to establish the most effective recruitment procedures with older women.