Dendrites from all genotypes grow and arborize over the course of three weeks.

<p>Confocal images of MAP2 immunolabeled neurons at 7 DIV (A–D) and 14 DIV (E–H). Magnification bar = 50 µm. These and similar preparations were traced in their entirety using Neurolucida. (I–L) Examples of such tracings are shown for 21 DIV neurons. Color-coding of dendrite branches corresponds to branch order. The maps shown are not to scale, but have been sized to fit the frame.</p

LRRK2-G2019S^OE negatively regulates dendritic growth and branching.

<p>Line graph (A) plots total dendritic length per neuron over time for neurons of all four genotypes. Data are shown as mean ± SEM. Table (B) shows results of Bonferroni's post-tests, indicating the sources of significant differences identified in a two-way ANOVA (see text). *p<0.05; **p<0.01; ***p<0.001, n.s.  =  not significant. Numbers in parentheses refer to DIV. Line graph (C) is an expanded view of results from the first week of development shown in (A), and the table (D) shows the post hoc analyses from this subset. Line graph (E) plots the total number of dendritic branch points for all genotypes and the table (F) shows the results of Bonferroni's post-tests. The line graph (G) is an expanded view of the data shown in (E) and the table (H) shows the post hoc analyses from the first week of development. At least 10 neurons from at least two culture preparations each (at least 20 neurons total) were traced and measured per genotype per time point assessed.</p

LRRK2-KO neurites show increased motility and those from LRRK2-WT^OE and LRRK2-G2019S^OE show decreased motility on PLL.

<p>Line graphs plot cumulative motility of axons (A) and dendrites (B) over 7.5 hours. Data shown are mean ± SEM. A one-way ANOVA indicates significant differences in motility for both axons and dendrites (see text) and the sources for those differences were identified using Bonferroni's post-tests, and are shown in tables (C) and (D) for axons and dendrites, respectively; *p<0.05; **p<0.01; ***p<0.001. Scatter plots of mean total growth (extension minus retraction ± SEM) of axons (E) and dendrites (F) at the end of imaging. For each genotype, 10–18 neurons were tracked from at least two different culture preparations, one axon per neuron and 2–4 dendritic endpoints. (G) Bright field images of axon extension over time in a KO neuron illustrate the highly dynamic extension and retraction observed. Times (t in h) are indicated at the top. Arrowheads mark the position of the axonal growth cone at t = 0. Squares mark the site of furthest extension of the axonal growth cone, which was observed at t = 6 h.</p

LRRK2 levels or mutation do not affect axonal development or branching.

<p>(A) Image of a 3 DIV LRRK2-WT^OE transgenic neuron immunolabeled for SMI-31 (magenta) and MAP2 (green) illustrates the normal polarization of proteins achieved in neurons from all genotypes. Scatter plots of polarity indices (PI) calculated for 3 DIV neurons of each genotype for SMI-31 (B) and for MAP2 (C). Mean ± SEM indicated by horizontal line and error bars. At least 10 neurons from two culture preparations each (at least 20 neurons total) were analyzed per genotype. Bar graphs of mean axon lengths (D) and mean number of axon terminal endings (E) measured in fixed 3 DIV neurons (± SEM). At least 10 neurons from at least two culture preparations each (at least 20 neurons total) were traced and measured per genotype.</p