An Elongator mouse model of ALS spotlights TDP-43 in the motor neuron nucleolus

Abstract

Dysfunction of Elongator is associated with amyotrophic lateral sclerosis (ALS). Here, we describe mouse models in which either Elongator subunit 1(Elp1) or subunit 3 (Elp3) is selectively ablated in alpha motor neurons of the spinal cord. These mice exhibit a progressive loss of motor strength and motor neuron degeneration. To interrogate the molecular mechanisms that contribute to motor neuron cell death in these mice, we examine multiple disease pathways, including the expression of TDP-43 whose cytoplasmic aggregation is associated with the human disease. Although TDP-43 is a well-characterized nuclear protein functioning in RNA metabolism and gene transcription, here we document TDP-43's robust presence in the nucleolus of wild-type motor neurons and its clearance from both the nucleus and the nucleolus of motor neurons in Elp conditional knockout mice. Thus, this study directly links dysfunction of Elongator with nucleolar disruption and TDP-43 clearing, two hallmark cellular pathologies of ALS.

Fig. 1. Expression of Elongator subunits in spinal cord motor neurons.

a–c E18.5. a, b An Elp1-LacZ reporter construct shows robust activity in the ventral horn of the spinal cord where alpha motor neurons are located. b Enlargement of the boxed region in (a). c In Chat-GFP embryos, GFP is present in both large diameter ventral horn neurons (≥ 440 μm², likely alpha motor neurons, arrows) and in smaller motor neurons (likely gamma motor neurons, arrowheads). d–f Adult (6 weeks). d, e Combination of Elp1-LacZ (black puncta) and Chat-GFP indicate that Elp1 is expressed in both large (d) and small (e) motor neurons. f Negative control. No puncta are present in motor neurons from Chat-GFP mice that lack the Elp1-LacZ cassette when incubated with x-gal substrate. g–i Spinal cord sections from 2-week-old Chat-GFP mice. IF using an anti-ELP3 antibody in Chat-GFP mice demonstrates that ELP3 is present in both large (arrows) and small (arrowheads) motor neurons. i Inset shows an enlargement of the boxed large diameter motor neuron. DRG dorsal root ganglion. Scale bar = 150 μm in (a), 75 μm in (c), 7.5 μm in (d–f), 100 μm in (g–i).

Fig. 2. Generation of cholinergic neuron-specific Elongator CKO mice.

a E16.5. In Rosa mTmG mice, GFP is produced in cell types expressing Cre recombinase. In Chat-Cre; Rosa mTmG mice, robust GFP expression is seen in large motor neurons in the ventral horn (arrows) and only sporadically seen in smaller motor neurons (arrowheads). b ELP1 protein levels are reduced in spinal cords of Elp1 CKO mice. Residual ELP1 in the CKO lanes corresponds to intact Elp1 expression in spinal cord cell types other than motor neurons. For uncropped images and size standards, see Fig. S2. c Masses of control and Elp1 CKO mice. No male data are included in the 12-week timepoint since males reach a humane endpoint at 10 weeks. Control 1, C (Chat-Cre; Elp1^+/LoxP); Control 2, C2 (Elp1^LoxP/LoxP). *P < 0.001, **P < 0.0001 (one-way ANOVA, Tukey’s HSD test). Bars = SD. For source data see Supplementary Data 1. d PaGE testing. Both male and female Elp1 and Elp3 CKOs begin to show motor function deficits during their first month and deficits increase as they age. C, control (Chat-Cre; Elp1 or Elp3 ^+/LoxP). n = five to seven animals per genotype and sex. Bars = SE. P values reach the ≤ 0.05 threshold at five weeks (Elp1 CKO females and Elp3 CKO males), six weeks (Elp1 CKO males), and seven weeks (Elp3 CKO females) (one-way ANOVA, Turkey’s HSD test). For individual data points and P values, see Supplementary Data 1. e–j 6 weeks. Both control (Chat-Cre; Elp1^+/LoxP) and CKO (Chat-Cre; Elp1^LoxP/LoxP) mice carry a single copy of Chat-GFP for visualization of motor neurons. ELP3 protein levels are reduced in large diameter (≥ 440 μm²), Chat-GFP-positive neurons of Elp1 CKO mice (h–j) compared to the control (e–g). Scale bar = 100 μm in (a), 25 μm in (e–j).

Fig. 3. Alpha motor neuron numbers and innervation of the NMJ in Elp1 CKO mice.

a–r Both control (Chat-Cre; Elp1^+/LoxP) and CKO (Chat-Cre; Elp1^LoxP/LoxP) mice carry a single copy of Chat-GFP for visualization of motor neurons. a–k Although both genotypes have equal numbers of large (≥ 440 μm²), motor neurons at E18.5, the numbers of large, NeuN-positive alpha motor neurons are dramatically diminished by six weeks in both male and female mice. E18.5, n = 4 C and 3 CKO; Adult, n = 3 male and 3 female mice for each genotype (* P < 0.05, ** P < 0.001, unpaired Student’s t-test. Bars = SD. See Supplementary Data 2 for source data. l–r The neuromuscular junction is progressively denervated in CKO mice. n = 3 female mice per genotype and time point. *P < 0.05 (4 weeks), **P < 0.001 (9 weeks), unpaired Student’s t-test. Bars = SD. m–r In nine-week-old control mice, motor neuron axons (GFP) completely innervate the NMJ (αB) (m–o) while in CKO mice (p–r), the majority are partially to completely denervated (arrow). αB alpha bungarotoxin. Scale bar = 40 μm in (b, c), 30 μm in (d, e, m–r), 15 μm in (f–k).

Fig. 4. TDP-43 levels are diminished in both the nucleolus and the nucleus of Elp1 CKO mice.

All fluorescence quantification was performed using female mice. a–c IF with anti-TDP-43 antibody 10782-2-AP (Proteintech) that recognizes the second RNA recognition motif (RRM2) shows robust levels of fluorescence in the nucleolus (arrowhead) of neurons and more moderate levels in the nucleus (arrow). d Nucleolar TDP-43 is present in large alpha motor neurons (arrows) and in smaller cells, likely interneurons and gamma motor neurons (arrowheads). e, f Nucleoli of Elp1 CKO alpha motor neurons exhibit clearing of TDP-43. g The average size of large (≥ 440 μm²) alpha motor neurons is normal in Elp1 CKO mice at two weeks of age, but is significantly diminished by six weeks. h Quantification of TDP-43 fluorescence per unit area in the nucleolus (No), the nucleus (Nu), and the cytoplasm (Cyto) at two and six weeks in female mice. i The nucleolar area occupied by TDP-43 is significantly diminished at both two and six weeks. j, k The nuclear area occupied by TDP-43 is stable at both two and six weeks, although the fluorescence levels are diminished at six weeks (h, k). l Although normal at two weeks, the cytoplasmic area occupied by TDP-43 is diminished at six weeks. m The average number of TDP-43-positive nucleoli per cell is reduced at six weeks of age. n–q The area occupied by fibrillarin (FBL) (n–p), and the fibrillarin fluorescence per unit area (q) are also reduced in alpha motor neurons of Elp1 CKO mice. *P < 0.05, **P < 0.01. All P values correspond to an unpaired Student’s t-test. All bars = SD. For source data, see Supplementary Data 3 (TDP-43), Supplementary Data 4 (FBL), Supplementary Data 5 (# of nucleoli). Au arbitrary units. Scale bar = 6 μm in (a–c, e, f, k); 36 μm in (d); 3 μm in (n, o).