The Ubiquitin-Proteasome Reporter GFPu Does Not Accumulate in Neurons of the R6/2 Transgenic Mouse Model of Huntington's Disease

Impairment of the ubiquitin-proteasome system (UPS) has long been considered an attractive hypothesis to explain the selective dysfunction and death of neurons in polyglutamine disorders such as Huntington's disease (HD). The fact that inclusion bodies in HD mouse models and patient brains are rich in ubiquitin and proteasome components suggests that the UPS may be hindered directly or indirectly by inclusion bodies or their misfolded monomeric or oligomeric precursors. However, studies into UPS function in various polyglutamine disease models have yielded conflicting results, suggesting mutant polyglutamine tracts may exert different effects on the UPS depending on protein context, expression level, subcellular localisation and cell-type. To investigate UPS function in a well-characterised mouse model of HD, we have crossed R6/2 HD mice with transgenic UPS reporter mice expressing the GFPu construct. The GFPu construct comprises GFP fused to a constitutive degradation signal (CL-1) that promotes its rapid degradation under conditions of a healthy UPS. Using a combination of immunoblot analysis, fluorescence and immunofluorescence microscopy studies, we found that steady-state GFPu levels were not detectably different between R6/2 and non-R6/2 brain. We observed no correlation between inclusion body formation and GFPu accumulation, suggesting no direct relationship between protein aggregation and global UPS inhibition in R6/2 mice. These findings suggest that while certain branches of the UPS can be impaired by mutant polyglutamine proteins, such proteins do not necessarily cause total blockade of UPS-dependent degradation. It is therefore likely that the relationship between mutant polyglutamine proteins and the UPS is more complex than originally anticipated

Expression of Mutant or Cytosolic PrP in Transgenic Mice and Cells Is Not Associated with Endoplasmic Reticulum Stress or Proteasome Dysfunction

The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the UbG76V-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases

Targeting Huntington’s disease through histone deacetylases

Huntington’s disease (HD) is a debilitating neurodegenerative condition with significant burdens on both patient and healthcare costs. Despite extensive research, treatment options for patients with this condition remain limited. Aberrant post-translational modification (PTM) of proteins is emerging as an important element in the pathogenesis of HD. These PTMs include acetylation, phosphorylation, methylation, sumoylation and ubiquitination. Several families of proteins are involved with the regulation of these PTMs. In this review, I discuss the current evidence linking aberrant PTMs and/or aberrant regulation of the cellular machinery regulating these PTMs to HD pathogenesis. Finally, I discuss the evidence suggesting that pharmacologically targeting one of these protein families the histone deacetylases may be of potential therapeutic benefit in the treatment of HD

N-terminal acetylation and replicative age affect proteasome localization and cell fitness during aging

Contains fulltext : 152512.pdf (publisher's version ) (Open Access)Specific degradation of proteins is essential for virtually all cellular processes and is carried out predominantly by the proteasome. The proteasome is important for clearance of damaged cellular proteins. Damaged proteins accumulate over time and excess damaged proteins can aggregate and induce the death of old cells. In yeast, the localization of the proteasome changes dramatically during aging, possibly in response to altered proteasome activity requirements. We followed two key parameters of this process: the distribution of proteasomes in nuclear and cytosolic compartments, and the formation of cytoplasmic aggregate-like structures called proteasome storage granules (PSGs). Whereas replicative young cells efficiently relocalized proteasomes from the nucleus to the cytoplasm and formed PSGs, replicative old cells were less efficient in relocalizing the proteasome and had less PSGs. By using a microscopy-based genome-wide screen, we identified genetic factors involved in these processes. Both relocalization of the proteasome and PSG formation were affected by two of the three N-acetylation complexes. These N-acetylation complexes also had different effects on the longevity of cells, indicating that each N-acetylation complex has different roles in proteasome location and aging

Muscular and glenohumeral changes in the shoulder after brachial plexus birth palsy: an MRI study in a rat model

<p>Abstract</p> <p>Background</p> <p>Shoulder abnormalities are the major cause of morbidity in upper brachial plexus birth palsy (BPBP). We developed a rat model of upper trunk BPBP and compared our findings to previously reported animal models and to clinical findings in humans.</p> <p>Methods</p> <p>Forty-three 5-day-old newborn rats underwent selective upper trunk neurectomy of the right brachial plexus and were studied 3 to 20 weeks after surgery. The passive shoulder external rotation was measured and the shoulder joint was assessed bilaterally by a 7.2T MRI bilaterally.</p> <p>Results</p> <p>We found a marked decrease in passive shoulder external rotation, associated with a severe subscapularis muscle atrophy and contracture. None however developed the typical pattern of glenohumeral dysplasia.</p> <p>Conclusions</p> <p>In contradiction with previous reports, our study shows that the rat model is not adequate for preclinical studies of shoulder dysplasia. However, it might serve as a useful model for studies analyzing shoulder contracture occurring after upper BPBP.</p

Muscular and glenohumeral changes in the shoulder after brachial plexus birth palsy : an MRI study in a rat model

Shoulder abnormalities are the major cause of morbidity in upper brachial plexus birth palsy (BPBP). We developed a rat model of upper trunk BPBP and compared our findings to previously reported animal models and to clinical findings in humans. Forty-three 5-day-old newborn rats underwent selective upper trunk neurectomy of the right brachial plexus and were studied 3 to 20 weeks after surgery. The passive shoulder external rotation was measured and the shoulder joint was assessed bilaterally by a 7.2T MRI bilaterally. We found a marked decrease in passive shoulder external rotation, associated with a severe subscapularis muscle atrophy and contracture. None however developed the typical pattern of glenohumeral dysplasia. In contradiction with previous reports, our study shows that the rat model is not adequate for preclinical studies of shoulder dysplasia. However, it might serve as a useful model for studies analyzing shoulder contracture occurring after upper BPBP

Recombination-induced tag exchange to track old and new proteins

The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods

A Fluorescent Broad-Spectrum Proteasome Inhibitor

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx3L3VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.