Developmental and maintenance defects in Rett syndrome neurons identified by a new mouse staging system in vitro

Abstract

Rett Syndrome (RTT) is a neurodevelopmental disorder associated with intellectual disability, mainly caused by loss-of-function mutations in the MECP2 gene. RTT brains display decreased neuronal size and dendritic arborization possibly caused by either a developmental failure or a deficit in the maintenance of dendritic arbor structure. To distinguish between these two hypotheses, the development of Mecp2-knockout mouse hippocampal neurons was analyzed in vitro. Since a staging system for the in vitro development of mouse neurons was lacking, mouse and rat hippocampal neurons development was compared between 1-15 days in vitro (DIV) leading to a 6-stage model for both species. Mecp2-knockout hippocampal neurons displayed reduced growth of dendritic branches from stage 4 (DIV4) onwards. At stages 5-6 (DIV9-15), synapse number was lowered in Mecp2-knockout neurons, suggesting increased synapse elimination. These results point to both a developmental and a maintenance setback affecting the final shape and function of neurons in RTT.

Keywords: Dendrites; Dendritic Spines; Neuronal morphology; Rett Syndrome; hippocampal neurons; neurodevelopment; staging system.

Figure 1

Morphological characterization of rat and mouse hippocampal neurons. (A) Neurons were counted from five sampling areas (rectangles) per coverslip. (B) DIV2 mouse hippocampal neuron immunostained for MAP2 (red) and Tau-1 (green) and quantification (mean + s.e.m.) of the percentage of neurons with one Tau-1 positive/MAP2-negative neurite in mouse and rat neuronal cultures. (Scale Bar = 10 μm) (C) Quantitative data of the average total dendritic length of mouse and rat hippocampal neurons in vitro. Data are expressed as mean ± s.e.m., n = 50. (D) Images of GFP-transfected rat and mouse hippocampal neurons at the indicated DIV (Scale Bar = 50 μm). (E) Number of primary dendrites, (F) branch points and (G) secondary dendrites in rat neurons at each DIV. (H) Primary dendrites, (I) branch points and (J) secondary dendrites in mouse neurons. The median line of the boxes refers to the 50 percentile of the entire population (N = 50 per DIV). Mann-Whitney Rank Sum Test, ^**P ≤ 0.01, ^*P ≤ 0.05.

Figure 2

Dendrites and spines maturation. (A) The length of the longest dendrite D1 (Lm) is divided by the average length of all dendrites D1, D2, D3, D(n) (Lsym). The dendritic arbor is polarized when the apical dendrite is specified, i.e., when Lm/Lsym = 2. (B) Percentage of neurons with polarized dendritic arbor in rat and mouse cultures at different DIV. Mann-Whitney Rank Sum Test, ^*P = 0.05. (C) Contribution of each dendrite to total dendritic length at DIV6 and DIV12, expressed as the ratio of measured dendrite length (Lm) to the Lsym. Dendrites are ranked longest to shortest (N = 50 neurons; 3–4 cultures). (D) Proximal and distal 40 μm-long fields of mouse and rat apical dendrites. (Scale Bar = 5 μm). (E) Spine density in apical dendrite proximal and distal region of mouse (dark gray) and rat (light gray) at DIV6, 9, 12, 15. (F,G) Spine type percentage (mean, standard errors). ^***p ≤ 0.001, ^**p ≤ 0.01, ^*p ≤ 0.05 (One-Way ANOVA with Holm-Sidak's pairwise multiple comparison).

Figure 3

Spine measurements and categorization at DIV6 and DIV12. Scatter plots representation for rat (A) and mouse (B) neurons. X-axis represents the length of each spine, while the y-axis shows the head diameter. Sorting of spine type was performed by operator during measurements; circle: stubby, triangle: thin and square: mushroom spines. (N = 15 fields from different neurons from 3–4 different cultures). (C) Average length (±SE) of thin spines and average (±SE) head diameter of mushroom spines at DIV6, 9 and 12 for mouse (dark gray bars) and rat (light gray bars) neurons. Student t-test ^*P ≤ 0.05.

Figure 4

Synapse characterization. (A) Representative image of SYN1 (red panel) and PSD95 (green panel) signals, and co-localization (merge panel) at DIV12 in rat and mouse hippocampal neurons (Scale bar = 5 μm). (B) Quantitative data representing the degree of co-localization between the two synaptic proteins Rat (gray) and Mouse (black) in proximal dendritic fields. In the Y-axes, the percentages of colocalized PSD95 with respect to the total PSD95 signals were plotted, while the X-axes reports the different time points considered as DIV. (C) Quantitative degree of co-localization between PSD95 and SYN1 in distal dendritic fields. (D) Representative image of SYN1 (red panel) and Gephyrin (green panel) signals, and co-localization (merge panel) at DIV12 in rat and mouse hippocampal neurons (E) Quantitative data representing the degree of co-localization between the two synaptic proteins Rat (gray) and Mouse (black) in proximal dendritic fields. In the Y-axes, the percentages of colocalized Gephyrin with respect to the total Gephyrin signals were plotted, while the X-axes reports the different time points considered as DIV. (F) Quantitative degree of co-localization between Gephyrin and SYN1 in distal dendritic fields. (G) Rat and (H) mouse spontaneous electrical activity at different DIV. ^**p ≤ 0.01 and ^*p ≤ 0.05 (One-Way ANOVA with Holm-Sidak's pairwise multiple comparison).

Figure 5

Morphological characterization of WT and Mecp2^−/y mouse developing hippocampal neurons. (A) Representative images of both WT and Mecp2^−/y mouse hippocampal neurons at DIV12. (Scale Bar = 50 μm) (B) Quantification of relative amount of polarized neurons in culture Data are expressed as mean ± SD, n = 4 cultures (C) Quantitative data of the average total dendritic length. Data are expressed as mean ± s.e.m., n = 40. Quantification of primary dendrites (D), branching points (E) and of the secondary dendrites (F) at DIV6, 9, 12, and 15. The median line of the boxes refers to the 50 percentile of the entire population. Mann-Whitney Rank Sum Test, ^***P ≤ 0.001, ^**P ≤ 0.01, ^*P ≤ 0.05.

Figure 6

Characterization of spines and synapses in WT and Mecp2^−/y mouse hippocampal neurons. (A) Proximal and distal fields of WT and Mecp2^−/y mouse apical dendrites. (Scale Bar = 5 μm) (B) Spine density in proximal and distal region of WT (black) and Mecp2^−/y (white) apical dendrites at DIV6, 9,12, 15. (C,D) Spine type percentage with respect to total spine number at different time points (mean + standard errors). (E) Synapse identification by SYN1 (red) and PSD95 (green) signals co-localization (merge) at DIV12 in WT and MeCP2^−/y neurons (Scale bar = 5 μm). (F) Quantitative analysis of SYN/PSD95 co-localization in WT (black) and Mecp2^−/y (white) in proximal (left panel) and distal dendritic fields (right panel). The Y-axis reports the percentages of colocalized PSD95 with respect to total PSD95 signals, while the X-axis reports the different DIV considered. (G) Synapse identification by SYN1 (red) and Gephyrin (green) signals co-localization (merge) at DIV12 in WT and MeCP2^−/y neurons (Scale bar = 5 μm). (H) Quantitative analysis of SYN/ Gephyrin co-localization in WT (black) and Mecp2^−/y (white) in proximal (left panel) and distal dendritic fields (right panel). The Y-axis reports the percentages of colocalized Gephyrin with respect to total Gephyrin signals, while the X-axis reports the different DIV considered. ^**p ≤ 0.01 and ^*p ≤ 0.05 (One-Way ANOVA with Holm-Sidak's pairwise multiple comparison).

Figure 7

Model of the in vitro neuron staging. Model of the development of rat and mouse hippocampal neurons in vitro, and the timing of developmental and maintenance failure in Mecp2^−/y neurons.