Inactivation of liver X receptor beta leads to adult-onset motor neuron degeneration in male mice

Abstract

Male mice with inactivated liver X receptor (LXR) beta suffer from adult-onset motor neuron degeneration. By 7 months of age, motor coordination is impaired, and this condition is associated with lipid accumulation and loss of motor neurons in the spinal cord, together with axonal atrophy and astrogliosis. Several of these features are reminiscent of the neuropathological signs of chronic motor neuron disease such as amyotrophic lateral sclerosis. Because the LXRs are important for cholesterol and lipid metabolism, we speculate that absence of LXRbeta leads to pathological accumulation of sterols and lipids that may themselves be neurotoxic or may modulate intracellular pathways and thereby predispose motor neurons to degeneration.

Fig. 1.

Box plot of rota-rod performance in 7-month-old mice. Five trials per day were given for 5 consecutive days. The retention time of mice on the rod was recorded, and mice staying for 90 sec were taken from the rod and recorded as 90 sec. White bars represent WT and striped bars represent LXRβ-/- (n = 10-13 for each group; box: mean ± SD; whisker: min, max; *, P < 0.01, Student's t test). (A) The LXRβ-/- male mice performed better for each day as the trial went on and they learned equally as well as the WT mice, but for the male LXRβ-/- mice, the retention time to stay on the rod was significantly shorter than for WT mice for all 5 days. (B) Female LXRβ-/- performed as well as the WT females, and both the learning ability and retention time to stay on the rod for all of the 5 days were normal.

Fig. 2.

Neuromuscular junction immunohistochemistry. (A and D) Motor endplates were identified with tetramethylrhodamine conjugated α-bungarotoxin (red). (B and E) Synapsin I, seen in green, was used as a marker for axon innervation. (C) Innervated endplate with yellow, indicating overlap of motor endplates with synapsin I staining. (F) Denervated endplates with no overlap of synapsin I staining. (Bar, 10 μm.)

Fig. 3.

Neuropathology in the lateroventral horns of L1 segments of the spinal cord in 7-month-old male mice. (A) Luxol fast blue was used to stain myelin, and counterstaining with Mayer's hematoxylin was performed to visualize cell nuclei. No evidence of demyelination was found in LXRβ-/- as compared with WT mice. However, loss of large α-motor neurons can be seen in the LXRβ-/- mice. Arrows indicate α-motor neurons in WT mice. The number of motor neurons was evaluated by counting large ChAT-positive cells and this count gave 6 ± 1 motor neurons per section for WT and 4 ± 1 motor neurons per section for LXRβ-/- mice, P < 0.001; Student's t test; n = 8 per genotype. Note that the picture only shows one of the two ventral horns in a section. (Bar, 50 μm.) (B) GFAP staining of astroglia can be seen as brown star-like shaped cell bodies, as indicated with arrows in LXRβ-/- sample. More activated astrocytes with large cell bodies can be seen in LXRβ-/- mice as compared with WT. Counting the number of GFAP-positive cells gave 12 ± 2 astrocytes per section for WT and 16 ± 3 astrocytes per section for LXRβ-/- mice; P = 0.004; Student's t test; n = 8 per genotype. Note that the picture only shows one of the two ventral horns in a section. (Bar, 50 μm.) (C) Lipid deposits were revealed by staining with Oil red O, as indicated by arrows. More and lager deposits can be seen in motor neurons of LXRβ-/- mice compared with WT mice; n = 5 per genotype. (Bar, 20 μm.)

Fig. 4.

Toluidine blue staining of L1 ventral nerve roots. The size of the axon calibers is smaller in LXRβ-/- mice as compared with WT, perhaps indicating axonal atrophy. Measurement of the mean diameter gave 4.1 ± 0.3 μm for WT and 3.7 ± 0.1 μm for LXRβ-/- mice; P = 0.022; Student's t test; n = 5 per genotype. (Insets) Higher magnification of selected parts can be seen. (Bar, 50 μm.)

Fig. 5.

Western blot of cerebellar extract from 7-month-old male mice. No difference in protein expression of either of the two calcium-buffering proteins, calretinin or calbindin, could be detected in LXRβ-/- mice as compared with WT. Size ladder is indicated on the left. α-tubulin (66 kDa) was used as a loading control (n = 3 for each genotype). (A) The protein expression of calretinin (≈30 kDa), mainly produced by granule cells in the cerebellum, did not differ in LXRβ-/- mice compared with WT. (B) The protein expression of calbindin (28 kDa), mainly produced by Purkinje cells in the cerebellum, did not differ in LXRβ-/- mice compared with WT.