Fig 1.
Diagram of the PrP:PLS3 construct.
A construct containing the mouse Prion (PrP) promoter, exon 1, intron 1 and part of exon 2 was fused to the human PLS3 cDNA. This same promoter was used previously to express SMN in neurons [38]. Arrows indicate the location of PrP exon1 forward primer and PLS3 reverse primer used to specifically amplify PLS3 transcripts produced by this transgene.
Fig 2.
Coding sequence of the plastin 3 transgene is functional.
(A) Lateral view of 28 hpf Tg(mnx1:GFP) showing ventrally extending motor axons of uninjected, smn morpholino (MO) injected, or smn MO + plastin 3 RNA (PLS3) injected embryos. (B) Embryos from three separate experiments (n = 19–24 embryos/experiment) were scored as having severe, moderate, mild, or no defects based on criteria in Carrel et al [45]. Mean ± SEM was plotted and significance was determined by two-tailed Mann-Whitney non-parametric rank test. *p = 0.0001, **p<0.0001.
Fig 3.
Expression of PrP:PLS3 transcript in brain and spinal cord.
(A) Endogenous mouse Pls3 expression in the brain (PLS-14: 6.7±0.8, PLS-39: 6.8±1.4, PLS-46: 7.4±1.4, control: 3.5±0.0 RFU, p = 0.197) and (B) spinal cord (PLS-14: 3.4±0.7, PLS-39: 9.5±0.8, PLS-46: 5.5±0.4, control: 5.4±0.0 RFU, p≤0.7) was measured using quantitative RT-qPCR. The difference in Pls3 expression between the lines was not statistically different from controls (ANOVA). (C) Human PLS3 expression in the brain (PLS-14:1057.9±168.8, p<0.001, PLS-39:1182.0±174.0 p<0.001, PLS-49:728.4±57.6 p = 0.005 vs. control 0±0.0 RFU) and (D) spinal cord (PLS-14: 1175.6±108.5 p<0.001, PLS-39: 852.3±96.7 p<0.001, PLS-46: 410.9±8.9 p<0.01 vs. control 0±0.0 RFU) is also shown for each transgenic line at P10. Note that there is a nearly 100-fold increase in expression of the PrP:PLS3 transgene the levels of endogenous mouse Pls in both brain (A,C) and spinal cord (B,D). (n = 5–7 animals for each transgenic line and tissue assayed). RFU is defined as Relative Fluorescent Units.
Fig 4.
Expression of PLS3 and endogenous mouse Pls3 in LCM isolated motor neurons.
Plastin expression was measured by quantitative RT-ddPCR in the motor neurons isolated from lumbar spinal cord tissue at P10 for each transgenic line (A) PLS3 expression is greatest in transgenic line PLS-14. (PLS-14: 170.5±45.3 p<0.05 vs. control, t-test, PLS-39: 151.0±57.7, PLS-46: 96.2±27.8, control: 0±0.0 RFU). Only PLS-14 expression was statistically different from control thus we pursued this line for protein analysis. PLS3 expression was not detected in the non-transgenic control motor neurons indicating the specificity of our primers. (B) Expression of mouse Pls is unchanged in the transgenic PrP:PLS3 lines when compared to a non-transgenic control. (PLS-14: 0.9±0.3, PLS-39: 1.4±0.2, PLS-46: 1.2±0.3, control: 1.1±0.1 RFU, not statistically significant from control.) Note that overexpression of human PLS3 is more than 100 fold greater than the amount of mouse Pls3 expression in the motor neuron. These results are similar to the expression assayed by qPCR in total spinal cord samples. (n = 3 mice for each transgenic line and control). RFU is defined as Relative Fluorescent Units.
Fig 5.
Total PLS3 Protein Expression in Spinal Cord tissue.
Total Plastin3 (PLS3 and mouse Pls) protein levels were measured in spinal cord extracts from P10 mice. While the level of PLS3 transcript is 100-fold increased, we found only a 2-fold increase in PLS3 protein in spinal cord samples. (A) Quantification was performed on 90μg, 45μg and 25μg of total protein with 3 independent samples for each protein concentration. A dilution series of total protein was loaded for each sample and the relative mean was determined by dividing the area under PLS3 peak by the area under the tubulin peak. (B) A representative 90μg, 45μg and 25μg serial dilution of one of the PrP:PLS3 samples from line PLS-14 and a non-transgenic control from the Western blot is shown. A total of 3 male mice were assayed for each protein concentration. All concentrations of PrP:PLS3 showed a statistically significant increase in PLS3 when compared to non-transgenic controls (t value 5.204 (90μg), 5.097 (45μg) and 5.086 (25μg) p<0.005) as determined by a one-way ANOVA.
Fig 6.
Weight of each PrP:PLS3 transgenic line in the presence and absence of mouse Smn.
Mice were weighted every day until weaning at 21 days of age. Each of the three PrP:PLS3 transgenic lines (PLS-14+/-, SMN2+/+; Smn-/-; Δ7SMN+/+, n = 22, average max. weight 3.9g), (PLS-39+/+, SMN2+/+; Smn-/-; Δ7SMN+/+, n = 20, average max. weight 3.5g), (PLS-46+/+, SMN2+/+; Smn-/-; Δ7SMN+/+, n = 32 average max. weight 3.7g) weigh slightly less than Δ7 SMA mice (SMN2+/+; Smn-/-; Δ7SMN+/+, n = 11, average max. weight 4.0g). There is no statistical difference in weight between SMA mice (Smn-/-) with or without the transgene.
Fig 7.
Survival is not increased in PrP:PLS3, Δ7SMA mice.
The Kaplan–Meier survival curve for three PrP:PLS3 transgenic lines (PLS-14+/-, SMN2+/+; Smn-/-; Δ7SMN+/+, n = 20), (PLS-39+/+, SMN2+/+; Smn-/-; Δ7SMN+/+, n = 20), (PLS-46+/+, SMN2+/+; Smn-/-; Δ7SMN+/+, n = 30) and SMA (SMN2+/+; Smn-/-; Δ7SMN+/+, n = 58) is shown. The median survival of PLS-14; Smn-/- (14.8±0.9 days, p = 33) and PLS-46; Smn-/- (13.6± 0.7 days, p = 0.05) was not statistically different from that of Δ7 SMA mice (Smn-/-) (15.7± 0.4 days). The survival of PLS-39, Smn-/- (13.4± 0.3 days, p<0.01) was statistically different from Δ7 SMA mice (Smn-/-) (15.7± 0.4 days) as the PLS-39 mice died on average 2 days before the SMA mice (Holm-Sidak pairwise comparison).
Fig 8.
Electrophysiology of PrP:PLS3 transgenic mice in the absence of Smn.
Analysis of the PLS-14 line in the presence and absence of Smn is compared (PLS-14+/-, SMN2+/+; Smn+/-; Δ7SMN+/+, compared to PLS-14+/-, SMN2+/+; Smn-/-; Δ7SMN+/+). Shown are bar graphs of endplate current (EPC), miniature endplate current (MEPC), quantal content, endplate current time constant (EPC time constant), and the degree of facilitation of the first EPC compared to the 10th EPC during the first 10 pulses of a 50 Hz simulation. All of the deficits in plastin SMA mice are similar in magnitude to the deficits we previously reported in the same line of Δ7 SMA mice. Control littermates (PLS-14+/-; SMN2+/+; Smn+/-; Δ7SMN+/+) = black bars, plastin SMA (PLS-14+/-; SMN2+/+; Smn-/-; Δ7SMN+/+) mice = grey bars. Error bars represent SEM. n = 4 mice and greater than 40 endplates for all data.