Castration restores function and neurofilament alterations of aged symptomatic males in a transgenic mouse model of spinal and bulbar muscular atrophy

Abstract

Transgenic models of neurodegenerative disease have proved uniquely powerful for delineating pathways of neuronal dysfunction and cell death. We have developed a transgenic model of the polyglutamine disease spinal and bulbar muscular atrophy (SBMA), an adult-onset, slowly progressive motor neuron disease caused by polyglutamine expansion in the androgen receptor (AR). Mice bearing a human AR with 112 glutamines reproduce many aspects of SBMA, including slowly progressive, gender-specific motor deficits, and neuronal intranuclear inclusions. Despite substantial motor deficits in male AR112Q mice, no motor neuron loss was observed, indicating that neuronal dysfunction, rather than neuronal death, is central to disease. Moreover, reduced levels of unphosphorylated neurofilament heavy chain (NF-H) were observed in motor neurons, suggesting a role for NF-H in SBMA neuronal dysfunction. The elimination of androgens by surgical castration of severely affected, aged 112Q male mice partially restored motor function as well as NF-H levels. These data suggest that hormone-based therapies designed to treat SBMA patients, even with advanced disease, are likely to be effective.

Figure 1.

AR protein expression in transgenic 112Q and 24Q transgenic male mice reveals detergent-soluble monomeric AR, as well as detergent-insoluble macromolecular complex in 112Q mice. Western blot of lysates from brain and spinal cord of 112Q, 24Q, and nontransgenic (ntg) male mice was hybridized with AR Ab N-20 (A). Lysate from AR112Q-expressing PC12 cells was used as a positive control. Western blot of lysates from brain of 112Q male and female, castrated (Cast.) 112Q male, 24Q male, and ntg male mice was hybridized with antibody N-20. Lysate from castrated 112Q male indicates a reduction in AR monomer as well as a decrease in insoluble macromolecular complex (B). Actin was used as a loading control.

Figure 2.

Male 112Q mice have more severe loss of motor function and earlier onset of deficiencies than their 112Q female littermates. 112Q males (n = 7) begin to show deficiencies in rotarod function beginning at 3 months of age; rotarod function continues to decline until ∼8 months of age, when 112Q mice are unable to stay on the rotarod (A). In contrast, 112Q females (n = 8) do not begin to show significant deficits in rotarod function until 8 months of age; although their rotarod function is impaired, 112Q females still retain the ability to stay on the rotarod, even at 12 months of age (A). 112Q Males also have decreased vertical activity, when compared with nontransgenic littermates (n = 8), whereas vertical activity in 112Q females is not significantly different from that of nontransgenic females (n = 12) (B); *p < 0.05. 112Q males have decreased grip strength with forepaws alone and all paws together, whereas female 112Q animals only show deficits in all paw grip strength (C), implying weakness of the hindlimbs. Error bars represent 1 SD. Male 112Q mice have an abnormal gait characterized by a widened, lower hindlimb posture and shorter hindlimb steps (D). The hindpaws were painted with blue paint, and the front paws were painted with red paint.

Figure 3.

NIIs are detected in 112Q male spinal cord. Shown are tissue sections from lumbar spinal cord. NIIs formed progressively in 112Q males from multiple, punctate inclusions at 6 weeks of age (C) to single, large inclusions by 4 months of age (A, B). Antibodies to N-terminal portions of the AR, such as AR-N20 (A) and AR-318 (B), detected NIIs, whereas C-terminal AR antibodies, such as AR-C19 (F) (12 months) and AR-441 (data not shown), did not. NIIs were not detected in spinal cord tissue from a nontransgenic male at 12 months of age (data not shown), in a 112Q female at 12 months of age (N20) (E), or in a 24Q male at 12 months of age (N20) (D).

Figure 4.

Unphosphorylated NF-H immunoreactivity is reduced in 112Q symptomatic males. Nissl staining reveals a similar number of motor neurons in the anterior horn of nontransgenic (A) and 112Q (B) males at 14 months of age, well after the onset of symptoms. Motor neuron size is also unaffected (G); however, immunostaining with SMI32, an Ab to unphosphorylated NF-H, reveals a reduction in both the number of immunoreactive neurons (p = 0.044) (H) and the intensity of staining (p = 0.038) (I) in 112Q males (D) when compared with age-matched nontransgenic littermates (C). The population of neurons detected with SMI32 did not differ significantly in size distribution (H). Error bars represent 1 SD. Quadriceps muscle from 14-month-old 112Q males (F) does not show signs of atrophy when compared with age-matched nontransgenic muscle (E).

Figure 6.

Castrated 112Q males show increased unphosphorylated NF-H immunoreactivity and decreased NII load. Aged (21 months) 112Q males demonstrated high NII load (E) in the spinal cord and a reduction of SMI32 immunoreactivity (D). Age-matched 24Q males had no NIIs (B) and normal amounts of SMI32 immunoreactivity (A), indicating that overexpression of AR is not responsible for this phenotype. Five months after castration, decreased NII load (H) and an increased number of SMI32-positive neurons (G) were observed in 23-month-old 112Q males (p < 0.05) (J). Error bars represent 1 SD. C, F, I, Merged images with the inclusion of DAPI stain.

Figure 5.

Castrated 112Q males show signs of recovery after surgery. Castration surgery was performed on a cohort of 9-month-old 112Q males. Three months after castration, rotarod function was significantly improved in castrated 112Q males (n = 9) when compared with intact 112Q males (n = 8), although still depressed in comparison with castrated nontransgenics (n = 8) (A). Error bars represent 1 SD. In a group of late-symptomatic 18-month-old males, rotarod function was severely depressed before castration. After surgery, two animals showed substantial improvement of rotarod function (B, C), whereas recovery was limited in the other two 112Q males (D, E).