Truncated Forms of the Androgen Receptor are Associated with Polyglutamine Expansion in X-Linked Spinal and Bulbar Muscular Atrophy

X-linked spinal and bulbar muscular atrophy (SBMA) isa rare form of motor neuron degeneration linked to aCAG repeat expansion in the first exon of the androgenreceptor gene coding for a polyglutamine tract. In orderto investigate the properties of the SBMA androgenreceptor in neuronal cells, cDNAs coding for a wild-type(19 CAG repeats) and a SBMA mutant androgen receptor(52 CAG repeats) were transfected into mouseneuroblastoma NB2a/d1 cells. The full length androgenreceptor proteins, of 110–112 kDa and 114–116 kDa forthe wild-type and mutant protein, respectively, weredetected by Western blotting in transfected cells. Inaddition, the presence of an expanded polyglutaminetract in the SBMA androgen receptor appears to enhancethe production of C-terminally truncated fragments ofthe protein. A 74 kDa fragment was particularlyprominent in cells expressing the SBMA androgenreceptor. From its size, it can be deduced that the 74 kDafragment lacks the hormone binding domain but retainsthe DNA binding domain. The 74 kDa fragment maytherefore be toxic to motor neurons by initiating thetranscription of specific genes in the absence ofhormonal control. Immunofluorescence microscopy ontransfected NB2a/d1 cells showed that, after hormoneactivation, the wild-type androgen receptor translocatedto the nucleus whereas the SBMA androgen receptorwas mainly localized in the cytoplasm in the form ofdense aggregates with very little androgen receptorprotein in the nucleus. This could explain the reductionin transcriptional activity of the SBMA mutant ascompared with wild-type androgen receptor.INTRODUCTIONX-linked spinal and bulbar muscular atrophy (SBMA), orKennedy’s disease, is a rare form of adult hereditary motorneuropathy characterized by progressive muscle weakness andatrophy, usually beginning in the third to fifth decade of life (1).The main pathological feature of SBMA is a marked loss andatrophy of lower motor neurons, particularly of the anterior horncells of the entire spinal cord (2–4). SBMA is associated with anexpansion of a polymorphic CAG repeat sequence in the firstexon of the androgen receptor gene coding for a polyglutaminetract in the N-terminal domain of the protein (5). In the normalpopulation, the size of the CAG repeat region ranges from 12 to30 repeats but, in patients with SBMA, the expanded domainvaries in size between 40 and 72 repeats (5–9).In addition to SBMA, a number of neurogenetic diseases arelinked to trinucleotide repeat expansion mutations in the codingregion of the gene involved. These include Huntington’s disease,spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy andMachado-Joseph disease. These are all slow progressive neuro-degenerative disorders affecting selective populations of neurons[for a review see (10)].The striking similarity of these mutations suggests a commonpathological mechanism leading to the modified protein havingacquired a neurotoxic function. Expanded polyglutaminestretches in proteins could modify the affinity for interactingproteins, as shown for huntingtin, the product of the Huntington’sdisease gene (11,12). Polyglutamine stretches have been shownto form stable -pleated sheet or ‘polar zippers’ which enableβprotein–protein interaction (13,14). When the polyglutaminerepeat tract expands beyond a certain number of glutamines itmay become an avid substrate for transglutaminases leading toprotein aggregates within the cell (15–17).The androgen receptor is expressed in diverse neuronalpopulations within the brain and spinal cord, but is preferentiallylocalized in motor nuclei of cranial nerves and in motor neuronsof the spinal cord (18–20). Androgens influence the survival offacial and hypoglossal motor neurons and increase the rate ofregeneration of their axons after axotomy (21–26). Androgensalso have a trophic effect on the sexually dimorphic neurons of thespinal nucleus of the bulbocavernosus (27,28).The capability of the SBMA mutant androgen receptor totransactivationally regulate gene expression in non-neuronal(29,30) or neuronal cells (31) is reduced in comparison with that