PMC

L1CAM–cytoskeleton interactions mediate retrograde and stationary behaviors. Bar graphs showing the percentage of trials engaging in stationary behavior, retrograde movement, or diffusion in cells expressing either wild-type or mutant forms of L1CAM. (A) Wild-type L1CAM treated with actin and microtubule inhibitors. In ND-7 cells expressing wild-type L1CAM, 28.6% of trials were stationary, 61.9% retrograde, and 23.8% diffused (n = 21). Cytochalasin D treatment (2 μM) eliminated retrograde movement (stationary 16.7%, retrograde 0%, diffusing 83.3%; n = 12). In contrast, nocadazole treatment (1 μM) inhibited retrograde movement slightly while leaving stationary behavior and diffusion unaffected (stationary 31.25%, retrograde 43.75%, diffusing 25%; n = 16). Treatment with DMSO alone at the same concentration used to dilute cytochalasin D and nocadazole caused a slight increase in retrograde movement (stationary 22.2%, retrograde 88.8%; diffusing 11.1%; n = 9). (B) L1CAM cytoplasmic tail mutants. In cells expressing an L1 mutant with an introduced stop codon designed to truncate the protein, leaving 4 aa of the predicted cytoplasmic tail (stop), the receptor diffused exclusively. In cells expressing the YF mutant of L1CAM (YF), 62.5% of trials were stationary and 37.5% were retrograde moving (56.2% diffused; n = 16). In cells expressing a histidine in place of Y1229 (FIGQY-H; YH, a mutation that inhibits ankyrin binding), 0% were stationary, 92.9% were retrograde-moving and 7.1% diffusing (n = 14). Finally, in cells expressing wild-type L1CAM treated with NGF to stimulate phosphorylation of Y1229, inhibiting ankyrin binding, 18.2% of trials displayed some form of stationary behavior, whereas 100% underwent retrograde transport on the cell surface (0% diffusing; n = 11).

Overexpression of full-length mutant huntingtin increases the number of stationary APP–YFP particles in primary striatal and hippocampal, but not cortical, neurons. A, Overexpression of mutant huntingtin does not affect velocity of either anterograde or retrograde APP–YFP movement in primary cortical neurons. Anterograde, 0.86 versus 0.67 μm/s, p > 0.05; retrograde, 0.69 versus 0.53 μm/s, p > 0.05. B, Overexpression of mutant huntingtin does not change the distribution APP–YFP moving particles in primary cortical neurons. C, Overexpression of mutant huntingtin does not affect velocity of either anterograde or retrograde APP–YFP movement in primary striatal neurons. Anterograde, 0.73 versus 0.74 μm/s, p > 0.05; retrograde, 0.47 versus 0.52 μm/s, p > 0.05. D, Overexpression of mutant huntingtin increases the number of stationary APP–YFP particles whereas it decreases the number of retrograde moving particles in primary striatal neurons. Retrograde, 33 versus 10%, p < 0.05; stationary, 31 versus 62%, p < 0.01. E, Overexpression of mutant huntingtin does not affect velocity of either anterograde or retrograde APP–YFP transport in primary hippocampal neurons. Anterograde, 0.65 versus 0.48 μm/s, p > 0.05; retrograde, 0.37 versus 0.26 μm/s, p > 0.05. F, Overexpression of mutant huntingtin increases the number of stationary APP–YFP particles whereas it decreases the number of anterograde moving particles in primary hippocampal neurons. Anterograde, 25 versus 11%, p < 0.05; stationary, 60 versus 82%, p < 0.01. N.S., Not significant. *p < 0.05; **p < 0.01. Error bars indicate SE.

FX2149 restored lysosomal transport in LRRK2-R1441C cells. SH-SY5Y cells co-transfected with GFP and either Flag-LRRK2-R1441C, Flag-LRRK2-WT, or vector plasmids at a 1:15 ratio were treated with FX2149 at 100 nM at 4 h post-transfection for 48 h. (A) Lysosomal events were quantified as stationary, anterograde, or retrograde and expressed as a percentage of total lysosomes for DMSO and FX2149 treatments. ^*p < 0.05 by ANOVA compared to vector control. {F_{(2, 7)} = 8.18; F_{(2, 7)} = 5.426; F_{(2, 7)} = 5.510 for stationary, anterograde, retrograde, respectively}. #p < 0.05 by t-test compared to R1441C with vehicle treatment. {F₍₄₎ = 2.18; F₍₄₎ = 1.399; F₍₄₎ = 1.778 for stationary, anterograde, retrograde, respectively}. (B) Quantification of total, anterograde, and retrograde run lengths of lysosomes traveling at least 2 microns in FX2149 treated and DMSO treated cells. ^*p < 0.05 by ANOVA compared to vector control. {F_{(2, 103)} = 4.249; F_{(2, 32)} = 0.4790; F_{(2, 79)} = 5.747 for stationary, anterograde, retrograde run length, respectively}. #p < 0.05 by t-test compared to R1441C with vehicle treatment. {F₍₃₆₎ = 2.003; F₍₁₀₎ = 2.705; F₍₂₄₎ = 1.500 for stationary, anterograde, retrograde run length, respectively}.

A GTP-binding inhibitor FX2149 improved mitochondrial transport impairment in LRRK2-R1441C cells. SH-SY5Y cells co-transfected with GFP and either Flag-LRRK2-R1441C, Flag-LRRK2-WT, or vector plasmids at a 1:15 ratio were treated with FX2149 at 100 nM at 4 h post-transfection for 48 h. (A) Mitochondria events were quantified as stationary, anterograde, or retrograde and expressed as a percentage of total mitochondria for DMSO and FX2149 treatments. ^*p < 0.05 by ANOVA compared to vector control. {F_{(2, 5)} = 15.49; F_{(2, 5)} = 10.32; F_{(2, 5)} = 22.64 for stationary, anterograde, retrograde, respectively}. #p < 0.05 by t-test compared to R1441C with vehicle treatment. {F₍₄₎ = 3.699; F₍₄₎ = 1.049; F₍₄₎ = 2.855 for stationary, anterograde, retrograde, respectively}. (B) Quantification of total, anterograde, and retrograde run lengths of mitochondria traveling at least 2 microns in FX2149 treated and DMSO treated GFP-expressing cells. ^*p < 0.05 by ANOVA compared to vector control. {F_{(2, 66)} = 3.774; F_{(2, 21)} = 0.1804; F_{(2, 42)} = 3.271 for stationary, anterograde, retrograde run length, respectively}. #p < 0.05 by t-test compared to R1441C with vehicle treatment. {F₍₂₄₎ = 4.974; F₍₆₎ = 3.828; F₍₁₆₎ = 6.340 for stationary, anterograde, retrograde run length, respectively}.