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Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle–regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Background and Purpose-Carnosine is a naturally occurring dipeptide with multiple neuroprotective properties. In addition, it is well tolerated in high doses with minimal side effects. The purposes of this study were to determine whether carnosine is neuroprotective in permanent focal cerebral ischemia and to determine potential mechanisms of neuroprotection. Methods-We investigated the efficacy of carnosine in a mouse model of permanent focal cerebral ischemia. The effects of carnosine were investigated with respect to neuronal damage and infarct formation, endogenous antioxidant status, and matrix metalloproteinase activity. Results-Carnosine significantly decreased infarct size and neuronal damage when administered at time points both before and after the induction of ischemia. Carnosine also decreased reactive oxygen species levels in the ischemic brain, preserved normal glutathione levels, and decreased matrix metalloproteinase protein levels and activity. Conclusions-Carnosine is neuroprotective in focal cerebral ischemia and appears to influence deleterious pathological processes that are activated after the onset of ischemia.

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Our studies of the Bombyx mori ecdysone receptor (BE) revealed that, unlike the Drosophila melanogaster ecdysone receptor (DE), treatment of BE with the ecdysone agonist tebufenozide stimulated high level transactivation in mammalian cells without adding an exogenous heterodimer partner. Gel mobility shift and transfection assays with both the ultraspiracle gene product (Usp) and retinoid X receptor heterodimer partners indicated that this property of BE stems from significantly augmented heterodimer complex formation and concomitant DNA binding. We have mapped this ''gain of function'' to determinants within the D and E domains of BE and demonstrated that, although the D domain determinant is sufficient for high affinity heterodimerization with Usp, both determinants are necessary for high affinity interaction with retinoid X receptor. Modified BE receptors alone used as replication-defective retroviruses potently stimulated separate ''reporter'' viruses in all cell types examined, suggesting that BE has potentially broad utility in the modulation of transgene expression in mammalian cells.Of the nuclear receptor superfamily members cloned and characterized from insect species, ecdysone receptor (EcR) is the only member for which an activating ligand is known (1, 2). EcR is also distinctive as the only characterized nonmammalian nuclear hormone receptor with no apparent mammalian homolog. Consequently, EcR continues to be studied for potential application to the modulation of transgene expression in mammalian cell types and transgenic animals (3, 4). The first study in which the potential of Drosophila melanogaster EcR (DE) was examined as a modulator of transgene expression revealed that DE required N-terminal fusion of a heterologous transactivating domain for high level function in mammalian cells and that, whereas 20-OH ecdysone was not able to efficiently stimulate DE transactivation, the ecdysteroid muristerone A (murA) was a potent activator (3). Subsequent studies of EcR biology indicated that ligand specificity was in large part determined by the obligate heterodimer partner available for EcR interaction. In insect cells, the natural heterodimer partner of EcR is the ultraspiracle gene product (Usp) (5), which together with EcR and ligand forms the functional ecdysteroid response complex (6-8). In mammalian cells, the retinoid X receptors (RXRs) were found to be capable of substituting for Usp (6-8), although there were several limitations. One limitation already described was that the range of activating ligands was essentially limited exclusively to murA (3,4,(6)(7)(8). A second limitation that took longer to fully define was that RXR (found in most, if not all, mammalian cell types) was a reluctant dimer partner of EcR, and very high endogenous levels were necessary for murA stimulation to occur (6-8). With the exception of 293 cells, DE could not support high level transactivation in the vast majority of mammalian cell types without superphysiological levels of RXR dimer partner supplied by...

Expanded polyglutamine (polyQ) tracts have been linked to a new class of human disease characterized by psychiatric/motor syndromes associated with specific patterns of neurodegeneration. We have used a direct viral approach to locally express expanded polyglutamine tracts fused to the green fluorescent protein (97Q-GFP) in the adult rat brain. We show that intrastriatal expression of 97Q-GFP causes the rapid formation of fibrillar, cytoplasmic, and ubiquitinated nuclear aggregates in neurons. 97Q-GFP expression also results in a specific temporal pattern of cell death in the striatum. Co-infection studies suggest that high level 97Q-GFP-expressing cells die during the first month, whereas low level 97Q-GFP-expressing neurons persist for up to 6 months after infection. These data indicate that cumulative expression of polyQ repeats throughout the life of the animal is not required to induce neuronal death, but rather acute overexpression of polyQ is toxic to adult neurons in vivo.

Carnosine (β-alanyl-L-histidine) has been shown to exhibit neuroprotection in rodent models of cerebral ischemia. In the present study, we further characterized the effects of carnosine treatment in a mouse model of permanent focal cerebral ischemia and compared them with its related peptides anserine and N-acetylated carnosine. We also evaluated the efficacy of bestatin, a carnosinase inhibitor, in ameliorating ischemic brain damage. Permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery (pMCAO). Mice were subsequently randomly assigned to receive an intraperitoneal injection of vehicle (0.9% saline), carnosine, N-acetyl carnosine, anserine, bestatin alone, or bestatin with carnosine. Infarct size was examined using 2,3,5-triphenyltetrazolium chloride staining 1, 3, and 7 days following pMCAO, and neurological function was evaluated using an 18-point-based scale. Brain levels of carnosine were measured in treated mice using high-performance liquid chromatography 1 day following pMCAO. We demonstrated that treatment with carnosine, but not its analogues, was able to significantly reduce infarct volume and improve neurological function compared with those in vehicle-treated mice. These beneficial effects were maintained for 7 days post-pMCAO. In contrast, compared with the vehicle-treated group, bestatin-treated mice displayed an increase in the severity of ischemic lesion, which was prevented by the addition of carnosine. These new data further characterize the neuroprotective effects of carnosine and suggest that carnosine may be an attractive candidate for testing as a stroke therapy.

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