T1736 is a novel phosphorylation site on the integrin β4 subunit that is phosphorylated downstream of protein kinase C and EGF receptor activation and is a substrate for protein kinase D1 in vitro and in cells. It contributes to the regulation of HD dynamics through modulating the association of β4 with plectin.

A major function of proteasomes and macroautophagy is to eliminate misfolded potentially toxic proteins. Mammalian proteasomes, however, cannot cleave polyglutamine (polyQ) sequences and seem to release polyQ-rich peptides. Puromycin-sensitive aminopeptidase (PSA) is the only cytosolic enzyme able to digest polyQ sequences. We tested whether PSA can protect against accumulation of polyQ fragments. In cultured cells, Drosophila and mouse muscles, PSA inhibition or knockdown increased aggregate content and toxicity of polyQ-expanded huntingtin exon 1. Conversely, PSA overexpression decreased aggregate content and toxicity. PSA inhibition also increased the levels of polyQ-expanded ataxin-3 as well as mutant α-synuclein and superoxide dismutase 1. These protective effects result from an unexpected ability of PSA to enhance macroautophagy. PSA overexpression increased, and PSA knockdown or inhibition reduced microtubule-associated protein 1 light chain 3-II (LC3-II) levels and the amount of protein degradation sensitive to inhibitors of lysosomal function and autophagy. Thus, by promoting autophagic protein clearance, PSA helps protect against accumulation of aggregation-prone proteins and proteotoxicity.

Several neurodegenerative disorders, including Huntington's disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein.Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.Supplementary material available online at

We developed single-image fluorescence lifetime imaging microscopy (siFLIM), a method for acquiring quantitative lifetime images from a single exposure. siFLIM takes advantage of a new generation of dedicated cameras that simultaneously record two 180°-phase-shifted images, and it allows for video-rate lifetime imaging with minimal phototoxicity and bleaching. siFLIM is also inherently immune to artifacts stemming from rapid cellular movements and signal transients.

SummaryAlzheimer's disease (AD) is hallmarked by amyloid-b (Ab) peptides accumulation and aggregation in extracellular plaques, preceded by intracellular accumulation. We examined whether intracellular Ab can be cleared by cytosolic peptidases and whether this capacity is affected during progression of sporadic AD (sAD) in humans and in the commonly used APPswePS1dE9 and 3xTg-AD mouse models. A quenched Ab peptide that becomes fluorescent upon degradation was used to screen for Ab-degrading cytoplasmic peptidases cleaving the aggregation-prone KLVFF region of the peptide. In addition, this quenched peptide was used to analyze Ab-degrading capacity in the hippocampus of sAD patients with different Braak stages as well as APPswePS1dE9 and 3xTg-AD mice. Insulin-degrading enzyme (IDE) was found to be the main peptidase that degrades cytoplasmic, monomeric Ab. Oligomerization of Ab prevents its clearance by IDE. Intriguingly, the Ab-degrading capacity decreases already during the earliest Braak stages of sAD, and this decline correlates with IDE protein levels, but not with mRNA levels. This suggests that decreased IDE levels could contribute to early sAD. In contrast to the human data, the commonly used APPswePS1dE9 and 3xTg-AD mouse models do not show altered Ab degradation and IDE levels with AD progression, raising doubts whether mouse models that overproduce Ab peptides are representative for human sAD.