Disease-Associated Mutant Ubiquitin Causes Proteasomal Impairment and Enhances the Toxicity of Protein Aggregates

Protein homeostasis is critical for cellular survival and its dysregulation has been implicated in Alzheimer's disease (AD) and other neurodegenerative disorders. Despite the growing appreciation of the pathogenic mechanisms involved in familial forms of AD, much less is known about the sporadic cases. Aggregates found in both familial and sporadic AD often include proteins other than those typically associated with the disease. One such protein is a mutant form of ubiquitin, UBB+1, a frameshift product generated by molecular misreading of a wild-type ubiquitin gene. UBB+1 has been associated with multiple disorders. UBB+1 cannot function as a ubiquitin molecule, and it is itself a substrate for degradation by the ubiquitin/proteasome system (UPS). Accumulation of UBB+1 impairs the proteasome system and enhances toxic protein aggregation, ultimately resulting in cell death. Here, we describe a novel model system to investigate how UBB+1 impairs UPS function and whether it plays a causal role in protein aggregation. We expressed a protein analogous to UBB+1 in yeast (Ubext) and demonstrated that it caused UPS impairment. Blocking ubiquitination of Ubext or weakening its interactions with other ubiquitin-processing proteins reduced the UPS impairment. Expression of Ubext altered the conjugation of wild-type ubiquitin to a UPS substrate. The expression of Ubext markedly enhanced cellular susceptibility to toxic protein aggregates but, surprisingly, did not induce or alter nontoxic protein aggregates in yeast. Taken together, these results suggest that Ubext interacts with more than one protein to elicit impairment of the UPS and affect protein aggregate toxicity. Furthermore, we suggest a model whereby chronic UPS impairment could inflict deleterious consequences on proper protein aggregate sequestration

The Origin and Nature of Tightly Clustered BTG1 Deletions in Precursor B-Cell Acute Lymphoblastic Leukemia Support a Model of Multiclonal Evolution

Recurrent submicroscopic deletions in genes affecting key cellular pathways are a hallmark of pediatric acute lymphoblastic leukemia (ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B-cell translocation gene 1 (BTG1), in a large cohort of pediatric ALL cases. BTG1 was found to be exclusively affected by genomic deletions, which were detected in 65 out of 722 B-cell precursor ALL (BCP-ALL) patient samples (9%), but not in 109 T-ALL cases. Eight different deletion sizes were identified, which all clustered at the telomeric site in a hotspot region within the second (and last) exon of the BTG1 gene, resulting in the expression of truncated BTG1 read-through transcripts. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism. Moreover, high levels of histone H3 lysine 4 trimethylation (H3K4me3), which is known to tether the RAG enzyme complex to DNA, were found within the BTG1 gene body in BCP-ALL cells, but not T-ALL cells. BTG1 deletions were rarely found in hyperdiploid BCP-ALLs, but were predominant in other cytogenetic subgroups, including the ETV6-RUNX1 and BCR-ABL1 positive BCP-ALL subgroups. Through sensitive PCR-based screening, we identified multiple additional BTG1 deletions at the subclonal level in BCP-ALL, with equal cytogenetic distribution which, in some cases, grew out into the major clone at relapse. Taken together, our results indicate that BTG1 deletions may act as “drivers” of leukemogenesis in specific BCP-ALL subgroups, in which they can arise independently in multiple subclones at sites that are prone to aberrant RAG1/RAG2-mediated recombination events. These findings provide further evidence for a complex and multiclonal evolution of ALL

Long-term proteasomal inhibition in transgenic mice by UBB+1 expression results in dysfunction of central respiration control reminiscent of brainstem neuropathology in Alzheimer patients

Aging and neurodegeneration are often accompanied by a functionally impaired ubiquitin–proteasome system (UPS). In tauopathies and polyglutamine diseases, a mutant form of ubiquitin B (UBB(+1)) accumulates in disease-specific aggregates. UBB(+1) mRNA is generated at low levels in vivo during transcription from the ubiquitin B locus by molecular misreading. The resulting mutant protein has been shown to inhibit proteasome function. To elucidate causative effects and neuropathological consequences of UBB(+1) accumulation, we used a UBB(+1) expressing transgenic mouse line that models UPS inhibition in neurons and exhibits behavioral phenotypes reminiscent of Alzheimer’s disease (AD). In order to reveal affected organs and functions, young and aged UBB(+1) transgenic mice were comprehensively phenotyped for more than 240 parameters. This revealed unexpected changes in spontaneous breathing patterns and an altered response to hypoxic conditions. Our findings point to a central dysfunction of respiratory regulation in transgenic mice in comparison to wild-type littermate mice. Accordingly, UBB(+1) was strongly expressed in brainstem regions of transgenic mice controlling respiration. These regions included, e.g., the medial part of the nucleus of the tractus solitarius and the lateral subdivisions of the parabrachial nucleus. In addition, UBB(+1) was also strongly expressed in these anatomical structures of AD patients (Braak stage #6) and was not expressed in non-demented controls. We conclude that long-term UPS inhibition due to UBB(+1) expression causes central breathing dysfunction in a transgenic mouse model of AD. The UBB(+1) expression pattern in humans is consistent with the contribution of bronchopneumonia as a cause of death in AD patients. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00401-012-1003-7) contains supplementary material, which is available to authorized users

Intestinal integrity and performance of broiler chickens fed a probiotic, a prebiotic, or an organic acid

This study aimed at evaluating the intestinal integrity, using light and scanning electron microscopy (SEM), and the performance of broiler chickens fed additives alternative to antimicrobials. A total of 1080 male chicks were distributed according to a completely randomized experimental design, with six treatments with six replicates of 30 birds each. The following treatments were evaluated: basal diet (control), basal diet supplemented with an antimicrobial, basal diet supplemented with a probiotic, basal diet supplemented with a prebiotic, basal diet with a symbiotic, and basal diet supplemented with organic acids. Weight gain, feed intake, feed conversion ratio and livability were recorded when broiler chickens were 10, 21, 35, and 42 days old. On day 42, 72 birds were individually weighed and sacrificed. In order to evaluate the morphometrics of the different intestinal wall layers, segments of the small intestine and the cecum were collected from two birds per replicate, and intestinal integrity (SEM) was evaluated in the same segments of two birds per treatment. During the starter period (1-21 days old), birds fed the alternative additives presented similar weight gain as those fed the antimicrobial product, but were not different from control birds. Feed conversion ratio of birds fed alternative additives was better than that of the control birds from one to 10 days of age, but not during the remaining rearing period, and was similar to the birds receiving the antimicrobial. The morphometric parameters of the different intestinal wall layers was not influenced by the treatments. During the total rearing period, the evaluated alternative additives did not improve intestinal integrity or broiler performance.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Sao Paulo State Univ, Sch Vet Med &Anim Sci, Dept Anim Prod, Botucatu, SP, BrazilSao Paulo State Univ, Dept Anat, Biosci Inst, Botucatu, SP, BrazilSao Paulo State Univ, Dept Vet Clin, Sch Vet Med &Anim Sci, Botucatu, SP, BrazilSao Paulo State Univ, Sch Vet Med &Anim Sci, Dept Anim Prod, Botucatu, SP, BrazilSao Paulo State Univ, Dept Anat, Biosci Inst, Botucatu, SP, BrazilSao Paulo State Univ, Dept Vet Clin, Sch Vet Med &Anim Sci, Botucatu, SP, Brazi