P. Thomas

We detected an interaction of the N-terminus of huntingtin (htt171) with the C-terminal region of the nuclear receptor co-repressor (N-CoR) using the yeast two-hybrid system. This interaction was repeat length dependent and specific to htt171; the co-repressor did not interact with the repeat carrying a section of atrophin 1 nor with the androgen receptor or polyglutamine alone. The interaction was confirmed using His-tagged Escherichia coli -expressed C-terminal human and rat co-repressor protein which pulled full-length huntingtin out of homogenized rat brain and in pull-down assays. The N-CoR represses transcription from sequence-specific ligand-activated receptors such as the retinoid X-thyroid hormone receptor dimers and other nuclear receptors including Mad-Max receptor dimers. The mechanism of this repression appears to be through the formation of a complex of repressor proteins including the N-CoR, mSin3 and histone deacetylases. We have used N-CoR and mSin3A antibodies in immunohistochemical studies and find that in Huntington's disease (HD) cortex and caudate, the cellular localization of these proteins is exclusively cytoplasmic whilst in control brain they are localized in the nucleus as well as the cytoplasm; mSin3A immunoreactivity also occurred in a subset of huntingtin positive intranuclear inclusions. The relocalization of repressor proteins in HD brain may alter transcription and be involved in the pathology of the disease.

X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease) is a polyglutamine (polyQ) disease in which the affected males suffer progressive motor neuron degeneration accompanied by signs of androgen insensitivity, such as gynecomastia and reduced fertility. SBMA is caused by CAG repeat expansions in the androgen receptor (AR) gene resulting in the production of AR protein with an extended glutamine tract. SBMA is one of nine polyQ diseases in which polyQ expansion is believed to impart a toxic gain-of-function effect upon the mutant protein, and initiate a cascade of events that culminate in neurodegeneration. However, whether loss of a disease protein's normal function concomitantly contributes to the neurodegeneration remains unanswered. To address this, we examined the role of normal AR function in SBMA by crossing a highly representative AR YAC transgenic mouse model with 100 glutamines (AR100) and a corresponding control (AR20) onto an AR null (testicular feminization; Tfm) background. Absence of endogenous AR protein in AR100Tfm mice had profound effects upon neuromuscular and endocrine-reproductive features of this SBMA mouse model, as AR100Tfm mice displayed accelerated neurodegeneration and severe androgen insensitivity in comparison to AR100 littermates. Reduction in size and number of androgen-sensitive motor neurons in the spinal cord of AR100Tfm mice underscored the importance of AR action for neuronal health and survival. Promoter-reporter assays confirmed that AR transactivation competence diminishes in a polyQ length-dependent fashion. Our studies indicate that SBMA disease pathogenesis, both in the nervous system and the periphery, involves two simultaneous pathways: gain-of-function misfolded protein toxicity and loss of normal protein function.

Endogenous opioids have been implicated in the pathway of tolerance to stresses. Hibernating tissues tolerate stress. Serum from hibernating woodchucks (hibernation induction trigger [HIT]), from summer nonhibernating animals (summer woodchuck active plasma [SWAP], and potential "hibernation opioid mimics" (D-Ala(2) D Leu(5) Enkephalin [DADLE]), and Deltorphin D (Delt D) were used as ischemic preconditioning agents (IPC) in an in vivo surgically induced cardiac ischemia rat model. Comparison of the IPC treatment was monitored by the molecular intensity of stress transcripts for polyubiquitin and HSP70 in Northern blot analyses. Delt D and HIT significantly reduced total polyubiquitin transcript expression, 2.1-fold and 1.4-fold, respectively, in ischemic tissue, while SWAP and DADLE did not differ from saline controls. The Delt D effect was sensitive to glibenclamide (Glb), a K(ATP) (potassium adenosine triphosphate) channel blocker. No inducible HSP70 was detected. The demonstration of an opioid IPC modulation of the ubiquitin stress pathway found here may be relevant for development of drug intervention in heart attacks and stroke.