β-amyloid precursor protein (APP) and its cleaved products are strongly implicated in Alzheimer’s Disease (AD). Endosomes are highly active APP processing sites and endosome anomalies associated with upregulated expression of early endosomal regulator, rab5, are the earliest known disease-specific neuronal response in AD. Here, we show that the rab5 effector APPL1 mediates rab5 over-activation in Down Syndrome (DS) and AD, which is caused by elevated levels of the β-cleaved carboxy-terminal fragment of APP (βCTF). βCTF recruits APPL1 to rab5 endosomes, where it stabilizes active GTP-rab5, leading to pathologically accelerated endocytosis, endosome swelling, and selectively impaired axonal transport of rab5 endosomes. In DS fibroblasts, APPL1 knockdown corrects these endosomal anomalies. βCTF levels are also elevated in Alzheimer brain, which is accompanied by abnormally high recruitment of APPL1 to rab5 endosomes as seen in DS fibroblasts. These studies indicate that persistent rab5 over-activation through βCTF-APPL1 interactions constitutes a novel APP-dependent pathogenic pathway in AD.

Kim and Ziff examine the molecular mechanism of synaptic scaling, showing that inhibition of neuronal excitability reduces calcium influx into neurons, resulting in decreased calcineurin activity. This leads to increased surface expression of calcium-permeable AMPA receptors as a homeostatic response.

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca 2+ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca 2+ -permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca 2+ -permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.LTP | Ca 2+ -permeable AMPA receptors | gene knockout | calcineurin

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Highlights d Pathological Rab5 activation in PA-Rab5 mice mimics AD-like endosomal dysfunction d PA-Rab5 mice have synaptic function/structure deficits and GSK-3b-mediated tauopathy d Rab5 overactivation in vivo underlies cholinergic degeneration and memory deficits d Endosomal dysfunction alone induces prodromal and degenerative AD-related changes

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A␤ peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.

In microfluidic filtration systems, one of the leading obstacles to efficient, continuous operation is clogging of the filters. Here, we introduce a lateral flow microfluidic sieving (μ-sieving) technique to overcome clogging and to allow continuous operation of filter based microfluidic separation. A low frequency mechanical oscillation was added to the fluid flow, which made possible the release of aggregated unwanted polystyrene (PS) particles trapped between the larger target PS particles in the filter demonstrating continuous μ-sieving operation. We achieved collection of the target PS particles with 100% separation efficiency. Also, on average, more than 98% of the filtered target particles were retrieved after the filtration showing high retrieval rates. Since the oscillation was applied to the fluid but not to the microfluidic filter system, mechanical stresses to the system was minimized and no additional fabrication procedures were necessary. We also applied the μ-sieving technique to the separation of cancer cells (MDA-MB-231) from whole blood and showed that the fluidic oscillations prevented the filters from being blocked by the filtered cancer cells allowing continuous microfluidic separation with high efficiency.

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