Localization of Hook1, Hook2 and Hook3 in relation to pathological markers in AD brain.

<p>(a) Hook1 detection in the hippocampus reveals strong labeling of neurofibrillary tangles (asterisk) and dystrophic neurites of neuritic plaques (+) co-labeled by AT8. Dystrophic neurites are detected more clearly by anti-Hook1 compared to AT8. (b) Hook2 protein is enriched in cellular components within amyloid plaques detected by anti-amyloid antibody 6F3D (+), but not overlapping with β-amyloid localization. Hook2 is not associated with tau aggregates. (c) Hook3 is expressed in neurons and labeling is increased in tangle-bearing neurons (asterisk) and dystrophic neurites (+) detected with AT8. Scale bar = 20 μm.</p

Immunohistochemical staining of Hook proteins in brain tissue of 6 month old P301L- tau transgenic mice.

<p>(a) Overview of Hook immunoreactivities in the prefrontal cortex (lateral of the central sulcus). Hook1 antibody shows a weak cytoplasmic immunoreactivity in the cortex, while Hook2 antibody marks distinct punctuate structures. Hook3 antibody clearly labels the soma of neurons. The staining shows a granular pattern. Note the tangle-like structures in the anti-Hook1 stained section (asterisk) (b) Staining of the brainstem with Hook1 and AT100 antibodies; tau-aggregates co-localise with Hook1. (c) Staining of the brainstem with Hook3 and AT100 antibodies. Hook3 antibody marks a neuron containing aggregated tau. (d) Staining of Hook2, GFAP and IBA1 in the brainstem; Hook2 co-localises with GFAP, but not with IBA1. Scale bars = a: 100 μm, b-d: 50 μm.</p

High specificity of Hook isoform-specific antibodies and Hook isoform localization in control brain and AD brain.

<p>(a) Affinity purified Hook isoform-specific antibodies were tested on lysates of N2A cells transfected with pEGFP-Hook plasmids. Lysates of N2A cells expressing EGFP-Hook1 (lane 1), EGFP-Hook2 (lane 2), EGFP-Hook3 (lane 3) and EGFP only (lane 4) were separated on 10% SDS-PAGE and transferred to PVDF membrane. The membrane was probed with 0.5 μg/ml anti-Hook1 antiserum (a-Hk1), 0.5 μg/ml anti-Hook2 antiserum (a-Hk2), 0.5 μg/ml anti-Hook3 antiserum (a-Hk3) or 0.25 μg/ml pan-Hook antiserum (a-pan Hk). Pan-Hook antiserum shows the expression of all EGFP-Hook fusion proteins in N2A cells (100–120 kDa), whereas the isoform-specific antisera only label the corresponding isoform. (b) No crossreactivity of affinity-purified Hook antibodies with PHF-tau prepared from AD brain was detected, phospho-tau antibody AT8 (lane1), a-Hook1 (lane 2), a-Hook2 (lane 3), a-Hook3 (lane 4). (c) The Dot blot shows that Hook2 antibody does not crossreact with β-amyloid; 100 ng EGFP-Hook2 N2A cell lysate (spot 1, 3); 100 ng β-amyloid 1–40 (spot 2, 4). (d) Immunohistochemical staining of the hippocampal CA3 region using Hook1, Hook2 and Hook3 antiserum in control brain and in AD brain (Braak stage V). Hook1 and Hook3 antibodies label tau pathology such as neurofibrillary tangles (asterisk) and dystrophic neurites in neuritic plaques (+). Granular neuronal Hook3 immunoreactivity present in control brains (arrowhead) is reduced in AD and NFT-associated Hook3 immunoreactivity is predominant. Amyloid plaque staining (+) is observed using the Hook2 isoform-specific antibody. Scale bar = 20 μm.</p

Hook protein levels in brain tissue of patients with AD.

<p>(a) Western blot analysis of the cytoplasmic fractions of brain homogenates. Grey substance of Brodmann area 22 (temporal cortex) was homogenized in detergent-free buffer and was subsequently ultracentrifuged. Twenty-three μg of protein of each supernatant were loaded on SDS-PAGE, analyzed by Western blot and probed with different antibodies. C: Braak stage 0, I: Braak stage I-III, V: Braak stage V-VI. (b) Densitometric analysis of protein immunoreactivity in brain homogenates of controls (Braak stage 0, n = 8), early stages of AD pathology (Braak stages I-III, n = 6) and late stages (Braak stages V-VI, n = 10). Data are means ± SEM. Statistical analysis was carried out with Kruskal-Wallis test and subsequently Mann-Whitney U test. * p < 0.05, *** p < 0.001 compared to control group with Mann-Whitney U test. OD: optical density, WB: Western blot. (c) Quantification of total Hook1 and total Hook3 in whole homogenates by ELISA. Whole homogenates comprising the soluble and the particulate fraction were prepared from tissue of Brodmann area 22 in detergent-free buffer. Hook proteins were captured from whole homogenates with isoform-specific Hook antibodies and detected with biotinylated pan-Hook antibody. Data were corrected by the negative control of the antibody background and groups were statistically tested with Kruskal-Wallis test and Mann-Whitney U test. Data are means ± SEM. (d) Association of Hook1 and Hook3 with PHF-tau analysed by a Sandwich ELISA. Hook proteins were captured from whole homogenates with anti-Hook1 or anti-Hook3 antibody. Detection with AT100 revealed an association of Hook1 and Hook3 with PHF-tau in four brain homogenates from patients with AD. (e) Hook3 binding to recombinant tau is increased by tau aggregation and not by tau phosphorylation. Recombinant Hook3 was incubated with 2N4R-tau either in its unmodified, aggregated (aggr.) or phosphorylated form (p-tau) or with both modifications and applied to a monoclonal pan-tau capturing antibody. Hook3 protein associated with captured tau protein was quantified using a polyclonal Hook3 antibody in this sandwich-ELISA (left panel). Hook3 immunoreactivity was corrected for background reactivity and equal binding of the different modified tau species was verified by detection with the polyclonal tau antibody (right panel).</p

Case details.

<p>Case details.</p

Effect of reduced Hook3 expression on endosomal transport and perinuclear clustering of EGF.

<p>HeLa cells were transfected with scrambled control RNA or one out of two Hook3 siRNAs and incubated for 72 h. (a) Knockdown of Hook protein levels. Cells were harvested 72 h after transfection and analyzed by SDS-PAGE and Western blotting. (b) Effect of Hook3 siRNA2 on endosomal trafficking of EGF. Cells were loaded with Alexa Fluor 555-EGF, incubated for the indicated intervals, fixed and analyzed by confocal microscopy. Hook3 siRNA slows down endosomal transport and perinuclear clustering of EGF indicated by less pronounced clustering of EGF-containing endosomes after 20 min and 90 min and an increased number of remaining fluorescent endosomes after 180 min of incubation. A similar result was obtained with Hook3 siRNA1. Scale bar = 20 μm. Cells are marked by cellular outlines. (c) Quantification of nuclear clustering by measuring the inhomogeneity of Alexa Fluor 555-EGF distribution inside the cell. Along a fluorescence intensity-profile a difference of fluorescence maximum and minimum was calculated. Mean and standard deviation are shown from four experiments (n = 4) using data from 15 cells each. Statistical significance was probed using Mann-Whitney U-Test * p< 0.05.</p

Hook3 knockdown increases production of endogenous β-amyloid in N2A cells.

<p>(a) N2A cells were transfected with two different siRNAs targeting Hook3 and with non-target siRNA as control (n = 3). Beta-amyloid accumulated for 48h in cell culture media was quantified by a murine amyloid-specific ELISA. Hook3 downregulation by two different siRNAs causes a 35% increase in secreted β-amyloid. * p < 0.05 compared to control group with student’s t-test. Data are means ± SD. (b) Western blot analysis of cell lysates demonstrate that transfection with Hook3 siRNA does not alter the levels of intracellular APP, BACE1 and PS1.</p

Effect of Hook3 siRNA on cytoplasmic transport complexes.

<p>N2A cells were transfected with control siRNA or one out of two Hook3 siRNAs and incubated for 72 h. (a) Knockdown of Hook protein levels. Cells were harvested 72 h after transfection and analyzed by SDS-PAGE and Western blotting. (b) Densitometric analysis of protein immunoreactivity. The averaged Hook3 immunoreactivity of Hook3 siRNA transfected cells is 24% and 15% of control level respectively. C: control RNA, 1: Hook3 siRNA1, 2: Hook3 siRNA2. <i>C</i>. Sedimentation analysis. Lysates of N2A cells transfected with control or Hook3 siRNA2 and sedimentation standards thyroglobulin (19S), katalase (13S) and BSA (3S) were subjected to 5–20% sedimentation analysis followed by SDS-PAGE and Western blotting. Knockdown of Hook3 shifts the presence of p150 to a protein complex smaller than dynactin, whereas between control and Hook3 siRNA treated cells no differences in DIC, p50 and KLC distribution were detected. Sucrose gradient and peak position of sedimentation standards are indicated at the top. (d) Western blot analysis of cell lysates transfected with control or Hook3 siRNA. Antibodies to DIC, KLC, p50, p150 and β-actin indicate no alteration in the total amount of these proteins by treatment with Hook3 siRNA2. A similar result was obtained with Hook3 siRNA1.</p

Cystatin F levels in cerebrospinal fluid from patients with Creutzfeldt-Jakob diseases, Alzheimer’s disease and controls.

<p>Cystatin F levels were measured in patients with autopsy-confirmed definitive Creutzfeldt-Jakob disease (Def CJD) or Alzheimer’s disease (Def AD), clinically diagnosed Alzheimer’s disease (Clin AD) and non-demented controls (Controls). <b>A</b> Age and gender distribution (****, p<0.0001 against all other categories, one-way ANOVA followed by Bonferroni’s Multiple Comparison test). <b>B</b> Sample storage of archival cerebrospinal fluid (CSF). <b>C</b> Time interval between lumbar puncture (LP) and <i>exitus</i> for deceased patients with autopsy-confirmed diagnosis of neurodegeneration (Def AD and Def CJD; **, p<0.01, Student’s t test). <b>D</b> Cystatin F levels in CSF from all study subjects from cohort 2 (*, p<0.05, one-way ANOVA followed by Bonferroni’s Multiple Comparison test). <b>E</b> Sub-analysis of cystatin F levels in CSF from Def CJD subjects according to the PrP type[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171923#pone.0171923.ref035" target="_blank">35</a>]. <b>F</b> Sub-analysis of cystatin F levels in CSF from Def CJD subjects according to the results of p14-3-3 Western blotting analysis as performed at the time of CSF sample collection. In all panels: Dots: denote individual subjects; bars: mean; error bars: standard deviation.</p

Cystatin F levels in brains from patients with Creutzfeldt-Jakob disease and Alzheimer’s disease.

<p><b>A</b> Cystatin F levels in frontal cortex (FCx) and cerebellum (Cb) of patients with autopsy-confirmed definitive Creutzfeldt-Jakob disease (Def CJD) or Alzheimer’s disease (Def AD). Dots: denote individual subjects; bars: mean; error bars: standard deviation. <b>B</b> Correlation of cystatin F levels in the two regions of each patient, denoted by a dot (R² 0.73 for Def AD and 0.83 for CJD, linear regression analysis). <b>C</b> Histologic analysis of cystatin F expression in frontal cortex of the same cohort of patients. Regions with abundant pathology, including amyloid β (Aβ) plaques and neurofibrillary tangles (Tau, further magnified in the inset) in Alzheimer’s disease and spongiosis and partially protease resistant prion protein (PrP) in Creutzfeldt-Jakob disease, from representative cases are depicted. Non-demented subjects were included as control (Ctrl). Lower row, left: encephalitis, with cystatin F positive infiltrating cells (further magnified in the inset at center). Lower row, right: bone marrow, with cystatin F positive cells (further magnified in the inset). Scale bar: 100 μm (black) or 25 μm (blue).</p