Figure 1

<p>Proteasome activities following lentiviral gene transfer of PA28γ and S5a, in control and HD fibroblasts. (A) Expression levels of gene and protein of S5a and PA28γ were determined using RT-PCR and Western blot after viral gene transfer to HD fibroblasts (lane 1 and 3; lenti-GFP transduced cells, lane 2; S5a transduced cells, and lane 4; PA28γ transduced cells). (B–G) Proteasome activities were increased by lentiviral gene transduction of PA28γ. Chymotrypsin-like (B, E), PGPH (C, F) and trypsin-like (D, G) activities were detected in normal control (<36 CAG) and HD patients' skin fibroblasts, which overexpress PA28γ (B–D) or S5a (E–G). The overexpression of PA28γ increased chymotrypsin and PGPH-like, but not trypsin proteasome activities in both normal control and HD fibroblasts compared to lenti-GFP transduction. Chymotrypsin activities and PGPH activities were increased in control fibroblasts by overexpression of S5a. However, in HD patients' fibroblasts, S5a did not increase PGPH activities and slightly decreased chymotrypsin-like activities (§, p<0.05 between control and HD fibroblasts. *, p<0.05 between the gene transferred groups of control protein GFP and PA28γ or S5a). The experiments were repeated three times in triplicate.</p

Figure 4

<p>Experimental exposure of PA28γ (A–D) or S5a (E–G) overexpressing control and HD model striatal neurons to toxin modeling pathophysiological processes observed in HD. (A) MG132 treated control (CTRL, 26 CAG) and HD model striatal neurons (Htt, 105 CAG) transduced with PA28γ. Shown are cell viabilities after 24 hours of exposure to MG132. (B–G) The reversible proteasome inhibitor, MG132 (B, E); the mitochondrial inhibitor, 3-NP (C, F); and the excitotoxin, QA (D, G) were used at various concentrations to treat control and HD model striatal neurons. HD model striatal neurons showed significantly decreased the resistance to those neuropathological toxins compared to control striatal neurons. PA28γ significantly improved cell survival, and S5a significantly decreased cell survival after exposure to MG132 and QA, but not 3-NP, respectively (§, p<0.05 between wild-type and mutant huntingtin overexpressing striatal neurons. *, p<0.05 between the gene transferred groups of control protein GFP and PA28γ or S5a). The experiments were repeated three times in triplicate.</p

Figure 2

<p>Lentiviral gene transfer of PA28γ and S5a, in control and HD model striatal neurons. (A) Schematic experimental outline of gene transfer and differentiation of striatal neurons followed by exposure to HD model experimental toxins. HD model striatal cells were gene engineered with PA28γ, S5a. After verification of expression for the transferred genes, cells were grown in medium containing 1μg/ml of doxycyclin for 48h before toxin treatment. After 24 h incubation in the toxic environment, medium was collected for the MTS assay and cells were harvested for proteasome activity determination. (B) Semiquantitative Western blot of the huntingtin showing a slight decrease of protein levels by lenti-viral transduction of PA28γ gene into HD model striatal cells. Results are shown as percentage of levels of lenti-GFP control group (* p<0.05).</p

Figure 3

<p>Proteasome activities following lentiviral gene transfer of PA28γ and S5a, in control and HD model striatal neurons. (A–F) Chymotrypsin-like (A, D), PGPH (B, E) and trypsin-like (C, F) activities were detected. Both wild type (CTRL, 26 CAG, control striatal neurons) and mutant huntingtin overexpressing HD model striatal neurons (Htt, 105 CAG, HD model striatal neurons) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000238#pone.0000238-Sipione1" target="_blank">[16]</a> were transduced with PA28γ or S5a. Basal proteasome activities were decreased in HD model striatal neurons compared to control cells with normal range of CAG repeats. However, the overexpression of PA28γ increased proteasome activities in both control and HD model striatal neurons. The overexpression of S5a decreased chymotrypsin-like activities in HD model striatal neurons and decreased PGPH activities in control striatal neurons. In contrast, total trypsin-like activities were slightly increased in both control and HD model striatal neurons after the gene transfer of S5a (§, p<0.05 between control and HD striatal neurons. *, p<0.05 between the gene transferred groups of control protein GFP and PA28γ or S5a). The experiments were repeated three times in triplicate.</p

p25/Cdk5-mediated phosphorylation of BACE1 increases its activity.

<p>A. Effect of <i>in vitro</i> p25/Cdk5-mediated phosphorylation of BACE1 on its enzymatic activity. B. Effect of a phosphorylation-defective BACE1 mutant (BACE1 T252A) on BACE activity in SK-N-BE(2)C cells. C. Effect of BACE1 T252A on the production of Aβ in HEK293 cells stably expressing APPsw. D. Effect of phosphorylation of Thr252 in BACE1 on basal hGH secretion in PC12 cells. Basal secretion from BACE1 or BACE1 T252A-transfected cells is plotted as a percentage of the secretion from PC12 cells transfected with the control plasmid. Representative immunoblot of extracts from transfected cells with an anti-His antibody is shown below the corresponding graph.</p

Phospho-BACE1 protein is elevated in the superior frontal cortex of human AD brains.

<p>A. Representative western blots of superior frontal cortex tissue (Brodmann area 9) from human AD patients (AD) and normal controls (Con). Arrowheads indicate phospho-BACE1 and the corresponding BACE1 dimer. B. Densitometric analysis of phospho-BACE1 and BACE1 signals in the western blot shown in A, normalized by β-actin signals. Amounts of phospho-BACE1 and BACE1 are plotted as a percent of the control. Control and AD groups consisted of 5 normal (mean age 65 ± 3 years; postmortem interval [PMI], 19 ± 1 h) and 10 AD patients (mean age 69 ± 2 years; PMI, 18 ± 2 h), respectively. *<i>p</i> < 0.05, **<i>p</i> < 0.01 versus the control by Mann-Whitney U test.</p

Phosphorylation of BACE1 at Thr252 by p25/Cdk5.

<p>A. Coomassie staining (left) and autoradiograph (middle) of SDS polyacrylamide gels containing the products of <i>in vitro</i> kinase assays that employed a BACE1 substrate and the p25/Cdk5 complex. Phospho-BACE1 and phospho-p25 are indicated by an arrow and an arrowhead, respectively. Histone H1, a known substrate of p25/Cdk5, was used as a positive control (right). B. Alignment of human, rat, and mouse BACE1 amino acid sequences. The consensus Cdk5 phosphorylation site is marked in bold. The phosphopeptide used to generate a phosphospecific BACE1 antibody is shown. C. Products of <i>in vitro</i> kinase assays that employed a BACE1 substrate and the p25/Cdk5 complex. Reactions were incubated for the indicated times (left panel) and treated with or without roscovitine, a Cdk5 inhibitor (right panel). Products were then analyzed by western blotting with phosphospecific BACE1 (P-BACE1) antibodies and anti-BACE1 antibodies. D. Western blots for detection of P-BACE1 and BACE1 from HEK293T cells transfected with plasmids encoding the indicated proteins. E. Rat brain lysates were analyzed by western blotting with anti-P-BACE1 antibodies. F. <b>Peptide competition assays demonstrated the specificity of anti-P-BACE1 antibodies.</b> Lysates from HEK293T cells transiently transfected with plasmids encoding p25 and Cdk5 (left panel) or mouse hippocampal lysates (right panel) were analyzed by western blotting with anti-P-BACE1 antibodies pre-incubated in the absence (None) or presence of BACE1-phosphopeptide (SLWYT(PO₄)PIRR), BACE1-nonphosphopeptide (SLWYTPIRR), or p25-phosphopeptide (SAGT(PO₄)PKRVI). G. Co-immunoprecipitation assays showed the interaction between BACE1 and Cdk5. HEK293T cell lysates that were transfected with plasmids encoding p25 and Cdk5 (left panel) or p25 TG mouse hippocampal lysates (right panel) were immunoprecipitated with control IgG or anti-Cdk5 antibodies and then subjected to immunoblot analysis with the indicated antibodies.</p

Phospho-BACE1 is markedly increased in the hippocampus of p25 transgenic (TG) mice.

<p>A. Immunohistochemistry of brain sections from p25 TG mice (20 weeks old, lower panel) and age-matched wild-type (WT) mice (upper panel) using anti-P-BACE1 antibodies. B. Representative western blots of indicated brain regions in two p25 TG mice and control littermates. Arrowheads indicate phospho-BACE1 and the corresponding BACE1 dimer. C. Densitometric analysis of western blots (normalized by β-actin signal) and BACE activity in p25 TG mice plotted as a percent of the WT. D. Aβ amounts in the hippocampus of p25 TG mice plotted as a percent of the WT. Data in C and D are means ± SEMs of 4–7 independent experiments. *<i>p</i> < 0.05 versus WT mice by 2-tailed Student’s t-test.</p

Phospho-BACE1 is cofractionated with p25 and endosome marker in an iodixanol gradient, and fraction 5 of p25 is a luminal protein.

<p>A. Nineteen-month-old p25 TG mouse hippocampus homogenates were separated through an iodixanol step gradient. Western blot analysis showed the distribution of the indicated proteins and organelle markers: Rab5 (early endosome), cathepsin D (lysosome), GM130 (Golgi apparatus), and Bip/Grp78 (ER). B. Two selected fractions, 5 and 10, were digested with PK in the presence or absence of the detergent Triton X-100 (TX-100) followed by immunoblotting analysis with indicated antibodies.</p

Additional file 1: Figure S1. of Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21WAF1/CIP1 expression

LRRK2 phosphorylates p53 and moesin at TXR sites in the in vitro kinase assay. A. Phosphorylation of various p53 proteins by the Flag-tagged full length LRRK2 protein (Invitrogen) after the in vitro kinase assay. Phosphorylated p53 was detected by either autoradiography or Western blot with the p-TXR antibody. B. Western blot analysis after the in vitro kinase assay using moesin, various GST-ΔN-LRRK2 WT and mutant proteins, and cold ATP. (DOC 88 kb