p120-catenin subfamily members have distinct as well as shared effects on dendrite morphology during neuron development in vitro

Abstract

Dendritic arborization is essential for proper neuronal connectivity and function. Conversely, abnormal dendrite morphology is associated with several neurological pathologies like Alzheimer's disease and schizophrenia. Among major intrinsic mechanisms that determine the extent of the dendritic arbor is cytoskeletal remodeling. Here, we characterize and compare the impact of the four proteins involved in cytoskeletal remodeling-vertebrate members of the p120-catenin subfamily-on neuronal dendrite morphology. In relation to each of their own distributions, we find that p120-catenin and delta-catenin are expressed at relatively higher proportions in growth cones compared to ARVCF-catenin and p0071-catenin; ARVCF-catenin is expressed at relatively high proportions in the nucleus; and all catenins are expressed in dendritic processes and the soma. Through altering the expression of each p120-subfamily catenin in neurons, we find that exogenous expression of either p120-catenin or delta-catenin correlates with increased dendritic length and branching, whereas their respective depletion decreases dendritic length and branching. While increasing ARVCF-catenin expression also increases dendritic length and branching, decreasing expression has no grossly observable morphological effect. Finally, increasing p0071-catenin expression increases dendritic branching, but not length, while decreasing expression decreases dendritic length and branching. These distinct localization patterns and morphological effects during neuron development suggest that these catenins have both shared and distinct roles in the context of dendrite morphogenesis.

Keywords: catenin; dendrite branching; dendrite morphogenesis; neuron morphology; p120-catenin subfamily.

FIGURE 1

Domains and structures of human p120-catenin subfamily proteins. Schematics on the left depict the domains of p120-catenin, delta-catenin, ARVCF-catenin, and p0071-catenin, with the corresponding predicted structure of each protein on the right. Data for domain schematics was obtained from UniProt, and models were made using AlphaFold (The UniProt Consortium, 2018; Jumper et al., 2021).

FIGURE 2

Endogenous localization of p120-subfamily catenins in primary neurons. Endogenous localization of (A) p120-catenin; (B) delta-catenin; (C) ARVCF-catenin; (D) p0071-catenin in primary rat hippocampal neurons at 1 DIV and 5 DIV. Relative fluorescent intensity in the soma growth cones, processes, and nucleus is shown below each example image for 1 DIV (left) and 5 DIV (right). Cells were co-stained with Phalloidin and DAPI to help visualize growth cones and nuclei for quantification, but for clarity these stains are not pictured in example images. Statistical significance was determined using a two-way Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons post-hoc test. Significance was assigned at P < 0.05 for *, and P < 0.0001 for ****, and ns for non-significant. N = 15 neurons from three biological replicates for each catenin at each DIV. Graph values show mean and standard deviation.

FIGURE 3

Morphological effects of p120-catenin expression in developing rat primary hippocampal neurons. Example images are shown of neurons that have been transfected with GFP, exogenous p120-catenin, and shRNA to knockdown p120-catenin. Cells were transfected at 3 DIV and analyzed at 7 DIV. These images have been masked to eliminate other cells in the visual field for clarity. Quantification of the average dendrite length per cell and average number of branch points per cell per each condition are shown below on the left, and Sholl analysis for each condition to the right. The number of cells analyzed in each condition were 35 for p120-catenin OE, 23 for p120-catenin KD, and 41 for GFP control. Samples were taken from five biological replicates. Statistical significance was determined using a two-way Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons post-hoc test. Significance was assigned at P < 0.05 for * and P < 0.0001 for ****. Chart values show mean and standard deviation.

FIGURE 4

Morphological effects of delta-catenin expression in developing rat primary hippocampal neurons. Example images are shown of neurons that have been transfected with GFP, exogenous delta-catenin, and shRNA to knockdown delta-catenin. Cells were transfected at 3 DIV and analyzed at 7 DIV. These images have been masked to eliminate other cells in the visual field for clarity. Quantification of the average dendrite length per cell and average number of branch points per cell per each condition are shown below on the left, and Sholl analysis for each condition to the right. The number of cells analyzed in each condition were 35 for delta-catenin OE, 23 for delta-catenin KD, and 41 for GFP control. Samples were taken from five biological replicates. Statistical significance was determined using a two-way Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons post-hoc test. Significance was assigned at P < 0.001 for *** and P < 0.0001 for ****. Chart values show mean and standard deviation.

FIGURE 5

Morphological effects of ARVCF-catenin expression in developing rat primary hippocampal neurons. Example images are shown of neurons that have been transfected with GFP, exogenous ARVCF-catenin, and shRNA to knockdown ARVCF-catenin. Cells were transfected at 3 DIV and analyzed at 7 DIV. These images have been masked to eliminate other cells in the visual field for clarity. Quantification of the average dendrite length per cell and average number of branch points per cell per each condition are shown below on the left, and Sholl analysis for each condition to the right. The number of cells analyzed in each condition were 46 for ARVCF-catenin OE, 17 for ARVCF-catenin KD, and 41 for GFP control. Samples were taken from five biological replicates. Statistical significance was determined using a two-way Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons post-hoc test. Significance was assigned at P < 0.001 for ***, P < 0.0001 for ****, and ns for non-significant. Chart values show mean and standard deviation.

FIGURE 6

Morphological effects of p0071-catenin expression in developing rat primary hippocampal neurons. Example images are shown of neurons that have been transfected with GFP, exogenous p0071-catenin, and shRNA to knockdown p0071-catenin. Cells were transfected at 3 DIV and analyzed at 7 DIV. These images have been masked to eliminate other cells in the visual field for clarity. Quantification of the average dendrite length per cell and average number of branch points per cell per each condition are shown below on the left, and Sholl analysis for each condition to the right. The number of cells analyzed in each condition were 32 for p0071-catenin OE, 27 for p0071-catenin KD, and 41 for GFP control. Samples were taken from five biological replicates. Statistical significance was determined using a two-way Brown-Forsythe and Welch ANOVA tests with Dunnett’s T3 multiple comparisons post-hoc test. Significance was assigned at P < 0.05 for *, P < 0.01 for **, P < 0.0001 for ****, and ns for non-significant. Chart values show mean and standard deviation.