6 research outputs found

    Predicted LRRK2 aggregation domains.

    No full text
    <p>(<b>A</b>) The PASTA algorithm by Trovato <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045149#pone.0045149-Trovato2" target="_blank">[19]</a> predicts an aggregation domain at 210–310 amino acid residues in the LRRK2 protein (NCBI accession number NP_940980). (<b>B</b>) An independent aggregation algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045149#pone.0045149-Tartaglia2" target="_blank">[21]</a> predicts two aggregation domains, one peak between amino acid residues (250 to 299) overlapping with the N-terminus region predicted by PASTA (A) and the other at amino acid residues 2050–2099. Tick marks in B represent sliding window of 50 amino acids.</p

    Increased N-term-LRRK2 aggregation in mesencephalic neurons.

    No full text
    <p>(<b>A</b>) Representative images showing N-term-LRRK2 cells with increased high intensity aggregates (arrows) in the cell body and neurites compared to WT-, C-term-, or N-del-LRRK2 constructs. Inset of the cell body is enlarged (bottom) to illustrate aggregates (arrows). Aggregates were quantitated utilizing number of granules/site (<b>B</b>), total granule area/site (<b>C</b>), and average granule intensity per site (<b>D</b>). Data shown are average of 3 independent experiments with more than 6 dishes/construct and 64 images/dish. (** P<0.01, ***P<0.001, one-way ANOVA with Bonferroni <i>post hoc</i> test). (<b>E</b>) Representative images showing the colocalization of EGFP-LRRK2 with MAP2 or TH immunostaining. Increased aggregates were observed in N-term-LRRK2 neurons including dopaminergic neurons. (<b>F</b>) Quantitation of numbers of LRRK2/MAP2 positive neurons with aggregates. Bars represent means of three experiments ± S.E.M. with multiple dishes and a total of about 600 MAP2 positive neurons analyzed. (*** P<0.001, one-way ANOVA with Bonferroni <i>post hoc</i> test). (<b>G</b>) Quantitation of numbers of LRRK2/TH-positives neurons with aggregates. Bars represent mean values from each of 4 experiments ± S.E.M. A total of about 150 dopaminergic neurons were analyzed (*** P<0.001, one-way ANOVA with Bonferroni <i>post hoc</i> test).</p

    Sequences Located within the N-Terminus of the PD-Linked LRRK2 Lead to Increased Aggregation and Attenuation of 6-Hydroxydopamine-Induced Cell Death

    Get PDF
    <div><p>Clinical symptoms of Parkinson's disease (PD) arise from the loss of substantia nigra neurons resulting in bradykinesia, rigidity, and tremor. Intracellular protein aggregates are a pathological hallmark of PD, but whether aggregates contribute to disease progression or represent a protective mechanism remains unknown. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been linked to PD in both familial cases and idiopathic cases and aggregates of the LRRK2 protein are present in postmortem PD brain samples. To determine whether LRRK2 contains a region of protein responsible for self-aggregation, two independent, bioinformatic algorithms were used to identify an N-terminal amino acid sequence as being aggregation-prone. Cells subsequently transfected with a construct containing this domain were found to have significantly increased protein aggregation compared to wild type protein or a construct containing only the last half of the molecule. Finally, in support of the hypothesis that aggregates represent a self-protection strategy, aggregated N-terminal LRRK2 constructs significantly attenuated cell death induced by the PD-mimetic, 6-hydroxydopamine (6-OHDA).</p> </div

    HA-tagged LRRK2 constructs show same pattern of aggregation.

    No full text
    <p>(<b>A</b>) SH-SY5Y cells were transfected with LRKK2 constructs and immunostained with anti-HA antibody after 16 hours. Arrows point to intracellular aggregates. (<b>B</b>) Quantitation of HA-LRRK2 cells with aggregates; Ninety-two percent of N-term-HA-LRRK2 cells showed aggregates compared to 49% of HA-WT-LRRK2 cells. Bars represent the average of the mean values from each of three experiments ± S.E.M. (***P<0.001, n = 3, Student's <i>t</i>-test).</p

    N-term-LRRK2 attenuates 6-OHDA-induced cell death.

    No full text
    <p>(<b>A</b>) Indicated LRRK2 construct was transfected into SH-SY5Y cells and the cell viability was assessed using PI staining after 72 hours. No significant cell death was detected for any construct. Bars represent mean values from each of three experiments ± S.E.M. (<b>B</b>) Cells were treated with 100 µM 6-OHDA 24 hours after transfection. Cell viability after 24 hours was assessed using the MTT reduction assay. Bars represent % survival versus untreated control. Bars represent mean values from each of 4 experiments ± S.E.M. (*P<0.05, n = 4, one-way ANOVA with Bonferroni <i>post hoc</i> test).</p

    LRRK2 N-terminal sequences show increased aggregation.

    No full text
    <p>(<b>A</b>) Schematic of WT, N-term, and C-term showing locations of functional groups, EGFP tag, and bioinformatically-determined N-terminal aggregation-prone region. N-terminal deleted LRRK2 was created by the removal of 35 amino acids including the aggregation region. (<b>B</b>) Western blots of cell lysates of SH-SY5Y cells transfected with indicated LRRK2 construct showed equal expression of the LRRK2 protein with anti-HA (top panel) or anti-C-terminal- (bottom panel) LRRK2 antibody. Equal lysate loading is indicated by β-actin loading control. (<b>C</b>) EGFP-LRRK2 (WT, N-term, C-term, or N-del) constructs were transfected into SH-SY5Y cells. Representative images after 24 hours show N-term-LRRK2 cells with increased aggregates as indicated by arrows. Quantitation of granule number (<b>D</b>), granule size (<b>E</b>), and granule intensity (<b>F</b>) over 24 hour time course using ImageXpress automated acquisition and analysis system. One hundred images were analyzed per construct across 4 wells. Line graphs show the average of the mean values from each of three experiments ± S.E.M., (*** denotes p value <0.001 for N-term versus WT, C-term or N-del- LRRK2 construct. <sup>###</sup> denotes p value <0.001, <sup>##</sup> denotes p value <0.01 for N-del versus WT, C-term or N-term-LRRK2 construct, n = 4, one-way ANOVA with Bonferroni <i>post hoc</i> test).</p
    corecore