SPIN90 Knockdown Attenuates the Formation and Movement of Endosomal Vesicles in the Early Stages of Epidermal Growth Factor Receptor Endocytosis

The finding that SPIN90 colocalizes with epidermal growth factor (EGF) in EEA1-positive endosomes prompted us to investigate the role of SPIN90 in endocytosis of the EGF receptor (EGFR). In the present study, we demonstrated that SPIN90 participates in the early stages of endocytosis, including vesicle formation and trafficking. Stable HeLa cells with knockdown of SPIN90 displayed significantly higher levels of surface EGFR than control cells. Analysis of the abundance and cellular distribution of EGFR via electron microscopy revealed that SPIN90 knockdown cells contain residual EGFR at cell membranes and fewer EGFR-containing endosomes, both features that reflect reduced endosome formation. The delayed early endosomal targeting capacity of SPIN90 knockdown cells led to increased EGFR stability, consistent with the observed accumulation of EGFR at the membrane. Small endosome sizes and reduced endosome formation in SPIN90 knockdown cells, observed using fluorescent confocal microscopy, strongly supported the involvement of SPIN90 in endocytosis of EGFR. Overexpression of SPIN90 variants, particularly the SH3, PRD, and CC (positions 643 - 722) domains, resulted in aberrant morphology of Rab5-positive endosomes (detected as small spots located near the cell membrane) and defects in endosomal movement. These findings clearly suggest that SPIN90 participates in the formation and movement of endosomes. Consistent with this, SPIN90 knockdown enhanced cell proliferation. The delay in EGFR endocytosis effectively increased the levels of endosomal EGFR, which triggered activation of ERK1/2 and cell proliferation via upregulation of cyclin D1. Collectively, our findings suggest that SPIN90 contributes to the formation and movement of endosomal vesicles, and modulates the stability of EGFR protein, which affects cell cycle progression via regulation of the activities of downstream proteins, such as ERK1/2, after EGF stimulation

Huntingtin’s spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function

The polyglutamine expansion in huntingtin protein causes Huntington’s disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single-particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical α-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases α-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease. DOI: http://dx.doi.org/10.7554/eLife.11184.00

Intracellular Cleavage of Amyloid β by a Viral Protease NIa Prevents Amyloid β-Mediated Cytotoxicity

<div><p>Nuclear inclusion a (NIa) of turnip mosaic virus is a cytosolic protease that cleaves amyloid β (Aβ) when heterologously overexpressed. Lentivirus-mediated expression of NIa in the brains of APP(sw)/PS1 mice significantly reduces cerebral Aβ levels and plaque depositions, and improves behavioral deficits. Here, the effects of NIa and neprilysin (NEP), a well-known Aβ-cleaving protease, on oligomeric Aβ-induced cell death were evaluated. NIa cleaved monomeric and oligomeric Aβ at a similar rate, whereas NEP only cleaved monomeric Aβ. Oligomeric Aβ-induced cytotoxicity and mitochondrial dysfunction were significantly ameliorated by NIa, but not by NEP. Endocytosed fluorescently-labeled Aβ localized to mitochondria, and this was significantly reduced by NIa, but not by NEP. These data suggest that NIa may exerts its protective roles by degrading Aβ and thus preventing mitochondrial deposition of Aβ.</p></div

NIa, but not NEP, restores Aβ-mediated mitochondrial dysfunction.

<p>Human neuroblastoma SH-SY5Y cells were transfected with pcDNA with no insert (Vec), or with pcDNA with cDNA encoding HA-NIa (NIa) or HA-NEP (NEP). After 24 h of incubation, cells were treated with 10 µM of oligomeric Aβ for an additional 48 h. (<b>A</b>) Cells were treated with 2.5 µM of JC-1, an indicator of Ψm, for 15 min at 37°C and visualized by confocal microscopy. The intensities of red and green JC-1 fluorescence were quantitated using the MetaMorph imaging software and their ratios were plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 7); *<i>p</i><0.05, **<i>p</i><0.01. (<b>B</b>) Cells were incubated with 30 µM of DHE, an indicator of ROS for 30 min. Low (panels a–d) and high (panels a’–d’) magnification images of cells were obtained using a confocal microscope. Data represent the number of fluorescent cells as a percentage of the total number of cells in the observed field. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 5); *<i>p</i><0.05, **<i>p</i><0.01.</p

Endocytosed oligomeric Aβ accumulates in lysosomes and mitochondria.

<p>Human neuroblastoma SH-SY5Y cells were treated with 2.5 µM of Alexa Fluor-labeled Aβ oligomers for 90 min (pulse) and were further incubated in fresh media for 90, 270, or 630 min (chase). Cells were co-stained with LysoTracker and MitoTracker, and observed under a confocal microscope. The images indicated by the open boxes are shown in a higher magnification in the adjacent columns. The yellow color in the merged image indicates co-localization of green (Alexa Fluor 488-labeled Aβ) and red (LysoTracker, Red/MitoTracker, Deep Red) fluorescence. The percentages of lysosomes (white bars) or mitochondria (gray bars) that co-localized with Aβ were plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 10). DIC, differential interference contrast.</p

In vitro cleavage of Aβ by NIa and NEP.

<p>For the cleavage assay, 2.5 µM of monomeric (<b>A</b>) and oligomeric Aβ (<b>B</b>) were incubated with 0.5 µM of purified NIa or NEP for 1, 2, and 3 h. The reaction mixture was separated on a PeptiGel (Elpis Biotech), blotted, and probed with the anti-Aβ 6E10 antibody. The densities of the intact Aβ bands were quantified using NIH ImageJ software and plotted. Each data point and error bar represents the mean ± SD (n = 3).</p

NIa, but not NEP, prevents accumulation of Aβ in mitochondria.

<p>Human neuroblastoma SH-SY5Y cells were transfected with pcDNA with no insert (Vec), or with pcDNA with cDNA encoding HA-NIa (NIa) or HA-NEP (NEP). After 24 h of incubation, cells were treated with 2.5 µM of Alexa Fluor 488-labeled Aβ oligomers for an additional 18 h. Cells were stained with LysoTracker (<b>A</b>) or MitoTracker (<b>B</b>), and observed by confocal microscopy. White arrowheads in panel A indicate Aβ that co-localized with lysosomes and open arrowheads in panel B indicate Aβ that co-localized with mitochondria. The percentages of lysosomes or mitochondria that co-localized with Aβ were plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 10); **<i>p</i><0.01.</p

NIa, but not NEP, prevents Aβ-mediated cytotoxicity.

<p>Human neuroblastoma SH-SY5Y cells were transfected with pcDNA with no insert (Vec), or with pcDNA with cDNA encoding HA-NIa (NIa) or HA-NEP (NEP). After 24 h of incubation, cells were treated with 10 µM of oligomeric Aβ for an additional 48 h. (<b>A</b>) Western blotting with an anti-HA antibody showed that the expression levels of NIa and NEP were similar. GAPDH (detected with an anti-GAPDH antibody) was used as the loading control. (<b>B</b>) Cell viability was determined by using the MTT assay. Each bar and error bar represents the mean ± SD (n = 3); *<i>p</i><0.05. (<b>C</b>) Cells were stained with Hoechst 33342 and viewed under a fluorescence microscope. Arrows indicate cells with pyknotic nuclei. The number of pyknotic nuclei was counted and plotted. Scale bar, 50 µm. Each bar and error bar represents the mean ± SD (n = 8); **<i>p</i><0.01.</p

Novel DNA Aptamers that Bind to Mutant Huntingtin and Modify Its Activity

The CAG repeat expansion that elongates the polyglutamine tract in huntingtin is the root genetic cause of Huntington’s disease (HD), a debilitating neurodegenerative disorder. This seemingly slight change to the primary amino acid sequence alters the physical structure of the mutant protein and alters its activity. We have identified a set of G-quadruplex-forming DNA aptamers (MS1, MS2, MS3, MS4) that bind mutant huntingtin proximal to lysines K2932/K2934 in the C-terminal CTD-II domain. Aptamer binding to mutant huntingtin abrogated the enhanced polycomb repressive complex 2 (PRC2) stimulatory activity conferred by the expanded polyglutamine tract. In HD, but not normal, neuronal progenitor cells (NPCs), MS3 aptamer co-localized with endogenous mutant huntingtin and was associated with significantly decreased PRC2 activity. Furthermore, MS3 transfection protected HD NPCs against starvation-dependent stress with increased ATP. Therefore, DNA aptamers can preferentially target mutant huntingtin and modulate a gain of function endowed by the elongated polyglutamine segment. These mutant huntingtin binding aptamers provide novel molecular tools for delineating the effects of the HD mutation and encourage mutant huntingtin structure-based approaches to therapeutic development

SPIN90 is a specific regulator of EGFR endocytosis.

<p><b>A.</b> (a) Whole-cell lysates of control and SPIN90 knockdown (KD) HeLa cells were separated using SDS-PAGE, and immunoblotted with polyclonal anti-EGFR antibody to quantify the total amounts of EGFR. The experiment was repeated more than three times. Levels of EGFR (b) and SPIN90 (c) were quantified via densitometry using MultiGauge software. <b>B.</b> The relative abundance of SPIN90 (a) and <i>EGFR</i> mRNA (b) was quantified in control and SPIN90 knockdown (KD) HeLa cells using RT-PCR (a) or qPCR (b). Values of qPCR for <i>EGFR</i> transcripts were normalized with those for <i>GAPDH</i> transcripts. <b>C.</b> Identification of the SPIN90 interaction with EGFR. Cells were subject to starvation for 16 h then treated with EGF (40 ng/ml) for 10 min. Cell lysate were immunoprecipitated with rabbit anti-IgG or rabbit anti-SPIN90 antibodies and blotted with rabbit anti-pY-EGFR antibody.</p